18 OR 19 Hydroxy prostaglandins

ABSTRACT

Novel 18 -, 19 - and 20 -hydroxy-prostaglandin derivatives of the formula I ##STR1## wherein the dotted line in the position 8-12 indicates the optional presence of a double bond, the waved lines in position 15 indicate that the hydroxyl group and the group R 4  are either in α- or β-position and Z represents a -- CH 2  CH 2  -- or a cis -- CH=CH -- group, and wherein R represents one of the groups: ##STR2## (wherein the waved lines indicate that the hydroxyl groups are either in α- or β-position and R 1  represents a hydrogen atom, a methyl or ethyl group), R 2  represents either an oxygen atom or a hydrogen atom and an α- or β-hydroxyl group, R 3  represents a hydrogen atom or a hydroxyl group and R 4  represents a hydrogen atom or a methyl group, with the proviso that when simultaneously, R 1 , R 3  and R 4  each represents a hydrogen atom, R 2  represents an oxygen atom, a double bond is in 8-12 position and the 15-hydroxyl group is in position α, R does not represent the group (b), but that when in addition to these conditions, Z represents a cis -- CH=CH -- group and the 8-12 position is saturated, R either represents the groups (b) or (c); and the pharmaceutically acceptable salts and esters thereof, novel process for their preparation by selective microbiological hydroxylation of compounds of formula II ##STR3## wherein the dotted line in the position 10-11 indicates the optional presence of a double bond in case the 8-12 position is saturated and the other symbols are as defined hereinabove, by means of microorganisms of the Division of Eumycota or, as far as the introduction of a hydroxyl group in the 18- or 19-position is concerned, of the Family of Streptomycetaceae, and, if desired, conversion of the 18 -, 19 - and 20 -hydroxy-prostaglandin derivatives thus obtained into pharmaceutically acceptable salts and esters thereof, and pharmaceutical compositions containing at least one of the novel hydroxy-prostaglandin derivatives of formula I.

STATE OF THE ART

Prostaglandins are members of a new hormonal system with a remarkablerange of biological and pharmaceutical properties. These compoundsbelong to a group of chemically related 20-carbon chain hydroxy fattyacids containing a five membered ring in the structure and differentdegress of unsaturation, a number of which have been reported in theliterature. For a review on prostaglandins and the definition of primaryprostaglandins, see for example, S. Bergstrom, Recent Progress inHormone Research, 22, pp. 153-175 (1966) and Science, 157, p. 382 ff(1967) by the same author (Karim, editor; "Prostaglandins: Progress inResearch" N.Y.-Wiley International (1972).

Prostaglandins are widely distributed in mammalian tissues and have beenisolated from natural sources in very small amounts. In addition, anumber of the naturally occurring prostaglandins have been prepared bychemical synthesis; note for example, J. Am. Chem. Soc., 91, p. 5675 ff(1969); J. Am. Chem. Soc., 92, p. 2586 ff (1970) and J. Am. Chem. Soc.,93, pages 1489-1493 (1971) and references cited therein; W. P. Schneideret al., J. Am. Chem. Soc., 90, p. 5895 ff (1968); U. Axen et al., Chem.Commun., p. 303 ff (1969) and W. P. Schneider, Chem. Commun., p. 304 ff(1969).

Because of the remarkable range of biological and pharmacologicalproperties exhibited by this family of compounds, a great deal ofinterest has focused upon such compounds, and the preparation of analogsof such compounds.

Microbiological conversions of prostaglandins or of prostaglandin-typecompounds have been described before, but these conversions usuallyrelate to the reduction of keto groups, mostly by bacteria or yeasts,for example the conversion of9,15-diketo-11-hydroxy-prosta-8(12),13(t)-dienoic acid by Flavobacteriumand Pseudomonas species into9-keto-11,15-dihydroxy-prosta-8(12),13(t)-dienoic acid (M. Miyano etal., Chem. Comm. (1971), 425).

U.S. Pat. No. 3,788,947 describes the fermentative reduction of the10(11) double bond in PGA-type prostaglandins, sometimes accompanied byconcomitant transformations, such as reduction of the 13(14) double bondor oxidation of the 15-hydroxyl group to a 15-oxo group. In oneparticular case, viz. reduction of the 10(11) double bond in9-keto-15α-hydroxy-prosta-5(c),10,13(t)-trienoic acid (PGA₂) withCunninghamella blakesleeana (ATCC 9245), there is described theconcurrent introduction of a 18-hydroxyl group.

The 19-hydroxyl derivatives of PGB₁(9-keto-15α-hydroxy-prosta-8(12),13(t)-dienoic acid) and PGB₂(9-keto-15α-hydroxy-prosta-5(c),8(12),13(t)-trienoic acid) are describedby S. Bergstrom, Science 157, p. 382 ff (1967).

OBJECTS OF THE INVENTION

It is an object of the invention to provide the novelhydroxy-prostaglandin derivatives of formula I shown below.

It is another object of the invention to provide a novel process for thepreparation of the hydroxy-prostaglandins of said formula I by selectivemicrobiological hydroxylation of compound of formula II shown below.##STR4##

It is a further object of the invention to provide pharmaceuticalcompositions for the treatment of bronchial asthma and otherbronchiospastic conditions, which comprise at least one of thehydroxy-prostaglandin derivatives of formula I, as well as a method forthe treatment of bronchial asthma or other bronchiospastic conditions byadministration of these pharmaceutical compositions.

THE INVENTION

The prostaglandin derivatives of the present invention are the new 18 -,19 - and 20 -hydroxy-prostaglandin derivatives of the formula I ##STR5##wherein the dotted line in the position 8-12 indicates the optionalpresence of a double bond, the waved lines in position 15 indicate thatthe hydroxyl group and the group R₄ are either in α- or β-position and Zrepresents a --CH₂ CH₂ --or a cis -- CH=CH -- group, and wherein Rrepresents one of the groups: (wherein the waved lines indicate that thehydroxyl groups are either in α- or β-position and R₁ represents ahydrogen atom, a methyl or ethyl group), R₂ represents either an oxygenatom or a hydrogen atom and an α- or β-hydroxyl group, R₃ represents ahydrogen atom or a hydroxyl group and R₄ represents a hydrogen atom or amethyl group, with the proviso that when simultaneously, R₁, R₃ and R₄each represents a hydrogen atom, R₂ represents an oxygen atom, a doublebond is in 8-12 position and the 15-hydroxyl grup is in position α, Rdoes not represent the group (b), but that when in addition to theseconditions, Z represents a cis -- CH=CH -- group and the 8-12 positionis saturated, R either represents the groups (b) or (c); and thepharmaceutically acceptable salts and esters thereof.

The present invention provides also a process for the selectivemicrobiologial introduction of a hydroxyl group in the 18-, 19- or20-position of prostaglandins and prostaglandin-type compounds, whichcomprises subjecting a compound of the general formula II, wherein thedotted line in the position 10-11 indicates the optional presence of adouble bond in case the 8-12 position is saturated and the other symbolsare as defined hereinabove, to the hydroxylation activity ofmicroorganism (or enzymes thereof) of the Division of Eumycota (Kingdomof Fungi) or, as far as the introduction of a hydroxyl group in the 18-or 19-position is concerned, of the Family of Streptomycetaceae (OrderActinomycetales, Class Schizomycetes, Division Protophyta of the Kingdomof Plants).

The 18 -, 19 - and 20 -hydroxy-prostaglandin derivatives thus obtainedcan be converted into pharmaceutically acceptable salts and estersthereof, by reacting the corresponding compound in the form of a freeacid with a suitable organic or inorganic base or ester-formingderivative.

Microbiological conversions of prostaglandins or of prostaglandin-typecompounds have been described before, but these conversions usuallyrelate to the reduction of keto groups, mostly by bacteria or yeasts,for example the conversion of9,15-diketo-11-hydroxy-prosta-8(12),13(t)-dienoic acid by Flavobacteriumand Pseudomonas species into9-keto-11,15-dihydroxy-prosta-8(12),13(t)-dienoic acid (M. Miyano etal., Chem. Comm. (1971), 425).

U.S. Pat. No. 3,788,947 describes the fermentative reduction of the10(11) double bond in PGA-type prostaglandins, sometimes accompanied byconcomitant transformations, such as reduction of the 13(14) double bondor oxidation of the 15-hydroxyl group to a 15-oxo group. In oneparticular case, viz. reduction of the 10(11) double bond in9-keto-15α-hydroxy-prosta-5(c),10,13(t)-trienoic acid (PGA₂) withCunninghamella blakesleeana (ATCC 9245), there is described theconcurrent introduction of a 18-hydroxyl group.

The 19-hydroxyl derivatives of PGB₁(9-keto-15α-hydroxy-prosta8(12),13(t)-dienoic acid) and PGB₂(9-keto-15α-hydroxy-prosta-5(c),8(12),13(t)-trienoic acid) are describedby S. Bergstrom, Science 157, p. 382 ff (1967).

The invention will be described with reference to the accompanyingdrawings wherein

FIG. 1 shows the reaction synthesis scheme for the preparation of thestarting materials for this invention from known starting materials;

FIG. 2 and FIG. 2 continued are the structural formula of the compoundsobtained as intermediates in the synthesis scheme shown in FIG. 1;

FIG. 3 shows the structural formula of the intermediates formed insynthesis of Compound A used as a starting material for the synthesisscheme of FIG. 1. The preparation of these intermediates A₆ to A₁ and Ais described in the Preparation section of the Specification and

FIG. 4 shows the structuctural formula of compound of Formula I of theinvention prepared as described from the compounds of Formula II and IIIalso shown.

The 18 -, 19 - and 20 -hydroxy-prostaglandin derivatives of formula Isupra are potent agents in the treatment of bronchial asthma and otherbronchospastic conditions. They have considerable relaxant activity onrespiratory smooth muscle, whereas they were found, in general, to bedevoid of appreciable activity on the intestinal and uterine smoothmuscle, as well as of appreciable irritant activity at the site ofapplication.

The utility of various prostaglandins and prostaglandin-derivativespresently in use in clinic is limited due to the occurrence ofundesirable side-effects, such as diarrhoea, abdominal cramps and/orirritation at the site of application.

The selective activity of the hydroxy-prostaglandin derivatives of thepresent invention was established by a multiparameter guinea-pig test.In this test guinea-pigs weighing 600-900 g are anaesthetized withsodium pentobarbitone (45 mg/kg, i.p.). Supplementary doses of sodiumpentobarbitone (3-6 mg i.v.) are administered when required (i.e. whenspontaneous respiration appears). The jugular vein is cannulated for theadministration of drugs. The guinea-pig is artificially respired with N₂O/O₂ (7/3), using a Keuskamp respirator.

Then the following functions are measured:

a. Blood pressure.

The common carotid artery is cannulated and the blood pressure measuredwith a pressure transducer.

b. Bronchial resistance and tracheal segment pressure.

A cannula is inserted into the trachea as close as possible to thethorax. The guinea-pig is artificially ventilated at 55 strokes/min. Thepresssure changes, assumed to be due to changes caused by thebronchioles, are measured by a pressure transducer attached to a sidearm of the cannula. The trachea is occluded at its lower end with ablind-ended cannula, while a cannula is further introduced into thetrachea as close as possible to the larynx. The system is completelyfilled with saline, and connected to a very sensitive pressuretransducer. Changes in the pressure measured (cm H₂ O) are assumed toreflect changes in the tone of the smooth muscle of the trachea. Thetrachea segment cannula is inserted with extreme caution so as to avoiddisruption of the nerve or blood supply to the segment.

c. Measurement of intestinal motility.

A balloon, containing distilled water and connected to a pressuretransducer, is inserted in the duodenum of the guinea-pig. Care is takenon ligaturing the cannula to avoid stricture of the duodenum. Theballoon is at a pressure of 10-20 mm Hg.

d. Measurement of uterine motility.

A polyethylene cannula is inserted into the uterus via the vagina to adepth of 2.5 cm. This is then tied off with a ligature around thecervix. The cannula is connected to a pressure transducer, the wholesystem being filled with liquid paraffin at a pressure of 10-20 mm Hg.

The present hydroxy-prostaglandin derivatives compare favourably in thismultiparameter test with well-known prostaglandins, such as PGF₂.sub.αand PGE₁, as is demonstrated for some compounds of this invention byTable 1.

                                      TABLE 1                                     __________________________________________________________________________    Guinea-pig multiparameter test.                                                                  TRACHEAL                                                                  DOSE                                                                              segment                                                                              BRONCHIAL                                                                            INTESTINAL                                                                           UTERINE                               COMPOUND       in μg                                                                          pressure                                                                             resistance                                                                           contractions                                                                         contractions                          __________________________________________________________________________    PGF.sub.2.sub.α                                                                         20 +      +      +      ++                                    PGE.sub.1       5  -      0      +      0                                     9-keto-15α,18{-dihydroxy-                                                              100 -      0      0      0                                     prost-13(t)-enoic acid                                                        9-keto-15α,19{-dihydroxy-                                                              100 -      0      0      0                                     prost-13(t)-enoic acid                                                        9β,15α,18{-trihydroxy-                                                            500 -0     0      0                                            prost-13(t)-enoic acid                                                        9β,15α,19{-trihydroxy-                                                            500 -      0      0      0                                     prost-13(t)-enoic acid                                                        9β,15α,20-trihydroxy-                                                             500 -      0      0      0                                     prost-13(t)-enoic acid                                                        __________________________________________________________________________

The activity of the 18 -, 19 - and 20 -hydroxy-prostaglandin derivativeson the respiratory tract musculature was further confirmed bydetermination of their ability to antagonize histamine-inducedbronchoconstriction. This test is a modification of the guinea-pigmultiparameter test, cannulations being carried out only for recordingblood pressure, tracheal segment pressure and bronchial resistance.

Histamine was injected i.v. in a dose of 4 μg (as base) at regularintervals throughout the experiment. If extra dosed of sodiumpentobarbitone had to be administered during the course of theexperiment to suppress voluntary respiration, the interval to the nextdose of histamine was lengthened.

Test compound were injected i.v. one minute before histamine in volumesless than 0.5 ml. The substances were washed in with 0.3 ml sterilesaline. The lungs were artificially over-ventilated one minute prior toinjection of the test compounds.

The ability of the compounds to counteract histamine-inducedbronchoconstriction and the increase in tracheal segment pressure wasdetermined using two dose levels -- a low one and a high one.

Some of the results obtained with compounds according to this invention,using PGE₁ as the reference compound, are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Antagonism of Histamine-induced Broncho-constriction                          (guinea-pig).                                                                                     DOSE     % INHIBITION                                     COMPOUND            in μg (± S.D.)                                      ______________________________________                                        PGE.sub.1           0.1      27.6 (± 10.5)                                                     1.0      53.2 (± 14.8)                                                     5.0      about 88                                         9-keto-15α,18{-dihydroxy-prost-                                                             1.0      25.9 (± 6)                                    13(t)-enoic acid    100      about 80                                         9-keto-15α,19{-dihydroxy-prost-                                                             1        23.9 (± 13.4)                                 13(t)-enoic acid    100      88.1 (± 5.0)                                  9β,15α,18{-trihydroxy-prost-                                                           100      28.1 (± 15.2)                                 13(t)-enoic acid                                                              19β,15α,19{-trihydroxy-prost-                                                          100      62.5 (± 6.6)                                  13(t)-enoic acid                                                              19β,15α,20-trihydroxy-prost-                                                           100      52.9 (± 17.9)                                 13(t)-enoic acid                                                              9α,15β,19{-trihydroxy-prost-                                                           500      about 60                                         13(t)-enoic acid                                                              9-keto-15β,19{-dihydroxy-prost-                                                              500      about 85                                         13(t)-enoic acid                                                              9-keto-15α,18{-dihydroxy-prost-                                                             1        45 (± 8.8)                                    5(c),13(t)-dienoic acid                                                       9-keto-15α,19{-dihydroxy-prosta-                                                            1        50.3 (± 4.3)                                  5(c),13(t)-dienoic acid                                                       9-keto-15α,20-dihydroxy-prosta-                                                             1        60.7 (± 14.6)                                 5(c),13(t)-dienoic acid                                                       9-keto-11α,15α,18{-trihydroxy-                                                        1        about 70                                         prost-13(t)-enoic acid                                                        9-keto-11α,15α,19{-trihydroxy-                                                        1        about 60                                         prost-13(t)-enoic acid                                                        9-keto-11α,15α,18{-trihydroxy-                                                        1        about 35                                         prosta-5(c),13(t)-dienoic acid                                                9-keto-11α,15α,19{-trihydroxy-                                                        1        about 70                                         prosta-5(c),13(t)-dienoic acid                                                ______________________________________                                    

The irritation at the site of application which is shown by variousprostaglandins and prostaglandin-derivatives can result in phlebitis atthe site of injection or in persistant coughing if (as in the case f.e.with PGE₁ and PGE₂) an aerosol is employed.

This effect can be studied using the Draize scoring method fordetermining irritation following topical application in the rabbit eye.PGE₁ was used as the reference compound; 1 μg/eye was the thresholdirritant dose with this compound; 5 μg was definitely irritant. Doses ofthe present hydroxy-prostaglandin derivatives which were equi-effectiveor more effective than PGE₁ against histamine-inducedbronchoconstriction, proved not to irritate the rabbit eye by topicalapplication. The results for some compounds of the invention are givenin Table 3.

                  TABLE 3                                                         ______________________________________                                                            DOSE                                                      COMPOUND            in μg IRRITATION                                       ______________________________________                                        PGE.sub.1           5        +                                                9-keto-15α,19{-dihydroxy-prost-                                                             100      -                                                13(t)-enoic acid                                                              9-keto-15α,20-dihydroxy-                                                                    100      -                                                prosta-5(c),13(t)-dienoic acid                                                9-keto-11α,15α,19{-trihydroxy-                                                        25       -                                                prost-13(t)-enoic acid                                                        9-keto-11α,15α,19{-trihydroxy-                                                        25       -                                                prosta-5(c),13(t)-dienoic acid                                                ______________________________________                                    

From the results obtained it may be concluded in view of theexplanations give above, that the 18 -, 1911- and 20 -prostaglandinderivatives of the present invention are particularly useful for thetreatment of bronchial asthma and other bronchospastic conditions. Theiradvantages over various of the presently available prostaglandinderivatives, are that they either have greater specificity (i.e. less orabsent activity on the intestines) or are less irritant at the site ofapplication, or both.

Specific new prostaglandin compounds of this invention are the 18 -,19 - and 20 -hydroxy derivatives of the following prostaglandins andprostaglandin-type compounds:

9-keto-15α-hydroxy-prosta-5(c),13(t)-dienoic acid;

9-keto-15α-hydroxy-prosta-5(c)8(12),13(t)-trienoic acid;

9-keto-11α,15α-dihydroxy-prost-13(t)-enoic acid;

9-keto-11α,15α-dihydroxy-prosta-5(c)-13(t)-dienoic acid;

9α,11α,15α-trihydroxy-prosta-5(c),13(t)-dienoic acid;

9-keto-15α-hydroxy-prost-13(t)-enoic acid;

9α,15α-dihydroxy-prost-13(t)-enoic acid;

9β,15α-dihydroxy-prost-13(t)-enoic acid;

9-keto-15β-hydroxy-prost-13(t)-enoic acid;

9-keto-15β -hydroxy-prost-13(t)-enoic acid;

9β,15β-dihydroxy-prost-13(t)-enoic acid;

9α,15α-dihydroxy-15β-methyl-prost-13(t)-enoic acid;

9α,15α-dihydroxy-20-ethyl-prost-13(t)-enoic acid;

9-keto-15α-hydroxy-15βmethyl 20-ethyl-prost-13(t)-enoic acid;

9-keto-15β-hydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid;

9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acid;

9α,15β-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid.

Specific prostaglandins and prostaglandin-type compounds of the generalformula II supra which can be microbiologically hydroxylated accordingto the process of this invention include:

9-keto-15α-hydroxy-prosta-5(c)-10,13(t)-trienoic acid;

9-keto-15α-hydroxy-prosta-5(c)8(12),13(t)-trienoic acid;

9-keto-11α,15α-dihydroxy-prost-13(t) -enoic acid;

9-keto-11α,15α-dihydroxy-prosta-5(c)13(t)-dienoic acid;

9α,11α,15α-trihydroxy-prost-13(t)-enoic acid;

9β,11α,15α-trihydroxy-prost-13(t)-enoic acid;

9α,11α,15α-trihydroxy-prosta-5(c),13(t)-dienoic acid;

9β,11α,15α-trihydroxy-prosta-5(c),13(t)-dienoic acid;

dl-9α,15α-dihydroxy-prost-13(t)-enoic acid;

dl-9β,15α-dihydroxy-prost-13(t)-enoic acid;

dl-9α,15β-dihydroxy-prost-13(t)-enoic acid;

dl-9β,15β-dihydroxy-prost-13(t)-enoic acid;

dl-9-keto-15α-hydroxy-prost-13(t)-enoic acid;

dl-9-keto-15β-hydroxy-prost-13(t)-enoic acid;

dl-9α,15α-dihydroxy-prosta-5(c),13(t)-denoic acid;

dl-9β,15α-dihydroxy-prosta-5(c),13(t)-dienoic acid;

dl-9α,15β-dihydroxy-prosta-5(c),13(t)-dienoic acid;

dl-9β,15β-dihydroxy-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15α-hydroxy-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15β-hydroxy-prosta-5(c),13(t)-dienoic acid;

dl-9α,15α-dihydroxy-15β-methyl-prost-13(t)-enoic acid;

dl-9α,15β-dihydroxy-15α-methyl-prost-13(t)-enoic acid;

dl-9β,15α-dihydroxy-15β-methyl-prost-13(t)-enoic acid;

dl-9β,15β-dihydroxy-15α-methyl-prost-13(t)-enoic acid;

dl-9-keto-15α-hydroxy-15β-methyl-prost-13(t)-enoic acid;

dl-9-keto-15β-hydroxy-15α-methyl-prost-13(t)-enoic acid;

dl-9α,15α-dihydroxy-15β-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15β-dihydroxy-15α-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15α-dihydroxy-15β-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15β-dihydroxy-15α-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15α-hydroxy-15β-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15β-hydroxy-15α-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15α-dihydroxy-20-methyl-prost-13(t)-enoic acid;

dl-9β,15α-dihydroxy-20-methyl-prost-13(t)-enoic acid;

dl-9α,15β-dihydroxy-20-methyl-prost-13(t)-enoic acid;

dl-9β,15β-dihydroxy-20-methyl-prost-13(t)-enoic acid;

dl-9-keto-15α-hydroxy-20-methyl-prost-13(t)-enoic acid;

dl-9-keto-15β-hydroxy-20-methyl-prost-13(t)-enoic acid;

dl-9α,15α-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15α-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15β-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15β-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15α-hydroxy-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15β-hydroxy-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15α-dihydroxy-20-ethyl-prost-13(t)-enoic acid;

dl-9β,15α-dihydroxy-20-ethyl-prost-13(t)-enoic acid;

dl-9α,15β-dihydroxy-20-ethyl-prost-13(t)-enoic acid;

dl-9β,15β-dihydroxy-20-ethyl-prost-13(t)-enoic acid;

dl-9-keto-15α-hydroxy-20-ethyl-prost-13(t)-enoic acid;

dl-9-keto-15β-hydroxy-20-ethyl-prost-13(t)-enoic acid;

dl-9α,15α-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15α-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9α ,15β-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15β -dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15α-hydroxy-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15β-hydroxy-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15α-dihydroxy-15β-methyl-20-methyl-prost-13(t)-enoic acid;

dl-9α,15β-dihydroxy-15α-methyl-20-methyl-prost-13(t)-enoic acid;

dl-9β,15α-dihydroxy-15β-methyl-20-methyl-prost-13(t)-enoic acid;

dl-9β,15β-dihydroxy-15α-methyl-20-methyl-prost-13(t)-enoic acid;

dl-9-keto-15α-hydroxy-15β-methyl-20-methyl-prost-13(t)-enoic acid;

dl-9-keto-15β-hydroxy-15α-methyl-20-methyl-prost-13(t)-enoic acid;

dl-9α,15α-dihydroxy-15β-methyl-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15β-dihydroxy-15α-methyl-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15α-dihydroxy-15β-methyl-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15β-dihydroxy-15α-methyl-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15α-hydroxy-15β-methyl-20-methyl-prosta-5(c),13(t)-dienoicacid;

dl-9-keto-15β-hydroxy-15α-methyl-20-methyl-prosta-5(c),13(t)-dienoicacid;

dl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acid;

dl-9α,15β-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid;

dl-9β,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acid;

dl-9β,15β-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid;

dl-9-keto-15α-hydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acidl

dl-9-keto-15β-hydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid;

dl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15β-dihydroxy-15α-methyl-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9β, 15α-dihydroxy-15β-methyl-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15β-dihydroxy-15α-methyl-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15α-hydroxy-15β-methyl-20-ethyl-prosta-5(c),13(t)-dienoicacid;

dl-9-keto-15β-hydroxy-15α-methyl-20-ethyl-prosta-5(c),13(t)-dienoicacid;

Some of the starting materials useful in preparing the novel 18 -, 19 -and 20 -hydroxy-prostagandin derivatives of general formula I supra areknown substances, such as:

9-keto-15α-hydroxy-prosta-5(c),10,13(t)-trienoic acid (PGA₂);

9-keto-15α-hydroxy-prosta-5(c),8(12),13(t)-trienoic acid (PGB₂);

9-keto-11α,15α-dihydroxy-prost-13(t)-enoic acid (PGE₁);

9-keto-11α,15α-dihydroxy-prosta-5(c),13(t)-dienoic acid (PGE₂);

9α,11α,15α-trihydroxy-prost-13(t)-enoic acid (PGF₁.sub.α);

9β,11α,15α-trihydroxy-prost-13(t)-enoic acid (PGF₁β);

9α,11α,15α-trihydroxy-prosta-5(c),13(t)-dienoic acid (PGF₂α);

9β,11α,15α-trihydroxy-prosta-5(c),13(t)-dienoic acid (PGF₂β).

Other starting materials in the process of this invention with theformula III ##STR6## wherein Z, R₁, R₂ and R₄ are as hereinbeforedefined, can be prepared according to the abbreviated schematic reactionsequence shown in FIG. 1, wherein each of the symbols A, B, IV and Vthrough IX represents compounds which may be depicted structurally bythe formulas shown in FIG. 2, wherein Z and R₁ are as hereinbeforedefined and the waved line in formula IV indicates a mixture of the α-and β-isomer.

The compounds of formula III (the free acids) are obtained by alkalinehydrolysis of the corresponding methyl esters of the formulas V, VI,VIII and IX shown in FIG. 2.

The compounds of formula A, wherein R₁ is as hereinbefore defined, whichare starting material in the reaction sequence shown in FIG. 2, areconveniently prepared according to the schematic overall reactionsequence shown in FIG. 3.

The compounds of formula A are prepared as follows: Step (a) is effectedby treating the compounds of formula A₆ with acetylene in the presenceof aluminium chloride at 0° C to yield the compounds of formula A₅. Thereaction is usually complete within four hours.

Step (b) is effected by treating the compounds of formula A₅ with sodiumiodide under anhydrous conditions and is typically conducted underreflux in acetone until the reaction is complete, usually from three totwelve hours, to obtain the compounds of formula A₄.

Step (c) is carried out by treating compounds of formula A₄ with [sodiumbis(2-methoxy ethoxy)aluminium hydride] and subsequently with an acid,e.g., sulfuric acid, at 0° C to obtain the compounds of formula A₃.

Step (d) is conveniently effected by treating the compounds of formulaA₃ with isopropenyl methyl ether in the presence of an acid catalyste.g., dichloroacetic acid or phosphorous oxychloride, at 0° C. Thecompound of formula A₂ wherein R₁ is a hydrogen atom, is also disclosedby Kluge et al., J. Am. Chem. Soc., 94, 7827 (1972).

Step (e) is effected by treating compounds of formula A₂ with t-butyllithium at -78° C to yield the compounds of formula A₁.

The last step of the above preparation, step (f), is convenientlyeffected by adding a solution of the compounds of formula A₁ to asolution of copper pentyne and hexamethyl phosphorous triamide to obtainthe compounds of formula A. The reaction is carried out at -78° C and isusually complete within one hour.

The compounds of formula IV are conveniently prepared by adding to thefreshly prepared compounds of formula A, the preparation of which isdescribed above, a compound of formula B, described by Bagli et al. inTetrahedron Letters, 465-470 (1966). The reaction is convenientlycarried out at -78° C and yields a mixture of two isomers of formula IV.

The compounds of formula V are conveniently prepared by removing theether protecting group by treating the above obtained mixture ofcompounds of formula IV with acetic acid at room temperature. Theresulting mixture of the compounds of formula V is separated into itsisomers (15α-OH and 15β-OH) by means of chromatography on silica gelusing ethyl acetate/hexane of increasing polarity as solvent.

The thus obtained compounds of formula V (15α-OH) are converted to amixture of the isomers of the compounds of formula VI (15α-OH, 9α-OH and15α-OH, 9β-OH) by treatment with sodium borohydride at 0° C. Thereaction is complete within about 45 minutes. The mixture of isomers isthen chromatographed on silica gel using ethyl acetate/hexane ofincreasing polarity as the solvent to obtain the compounds of formula VI(15α-OH, 9α-OH and 15α-OH, 9β-OH). Tn a similar manner theabove-obtained compounds of formula V (15β-OH) are converted into theindividual isomers, the compounds of formula VI (15β-OH, 9α-OH and15β-OH, 9β-OH). The thus obtained compounds of formula VI (15α-OH, 9α-OHor 15β-OH, 9α-OH) are treated with dichlorodicyano quinone for 36 hoursat room temperature in a benzene solution to yield the compounds offormula VII ( 9α-OH). Similarly, substituting the compounds of formulaVI (15α-OH, 9β-OH or 15β-OH, 9β-OH) for the compounds of formula VI(15α-OH, 9α-OH) yields the compounds of formula VII (9β-OH).

Treatment of the compounds of formula VII (9α-OH) with methylmagnesiumbromide in tetrahydrofuran at -30° C for 45 minutes yields a mixture ofcompounds of formula VIII (9α-OH, 15α-OH, 15β-CH₃ and 9α-OH, 15β-OH,15α-CH₃), which are separated into individual isomers by chromatographyon silica gel using ethyl acetate/hexane of increasing polarity assolvent. Substituting the compounds of formula VII (9β-OH) for VII(9α-OH) in the above reaction yields the compounds of formula VIII(9β-OH, 15α-OH, 15β-CH₃ and 9β-OH, 15β-OH, 15α-CH₃).

The thus obtained compounds of formula VIII (9α-OH, 15α-OH, 15β-CH₃) aretreated with a suspension of Celite (diatomaceous earth) and chromiumtrioxide in anhydrous methylene chloride under nitrogen in the presenceof pyridine for about one hour to yield the compounds of formula IX(15α-OH, 15β-CH₃). The latter compound is also obtained by substitutingthe compounds of formula VIII (9β-OH, 15α-OH, 15β-CH₃) for the compoundsof formula VIII (9α-OH, 15α-OH, 15β-CH₃). Substituting the compounds offormula VIII (9α-OH, 15β-OH, 15α-CH₃ or 9β-OH, 15β-OH, 15α-CH₃) for thecompounds of formula VIII (9α-OH, 15α-OH, 15β-CH₃)yields the compoundsof formula IX (15β-OH, 15α-CH₃).

The compounds of formulas V, VI, VIII and IX are converted to theircorresponding free acids by treatment with base, e.g., potassiumhydroxide at room temperature for about 2 hours, to yield the compoundsof formula III.

PREPARATION 1

This preparation illustrates methods for preparingdl-1-pentynyl-1-[trans-3-(2,2-methoxypropoxy)-1-decenyl] cuprate. (A, R₁=C₂ H₅)

a. A solution of 200 ml of octanoyl chloride (A₆, R₁ =C₂ H₅) in 750 mlof carbon tetrachloride is cooled on an ice bath and treated with 214 gof aluminium chloride in three portions over a 1 hour period whileacetylene is bubbled through the solution. The ice bath is removed andthe reaction mixture stirred at room temperature for 3 hours withadditional acetylene being added. At the end of this period, thereaction mixture is poured into 4 kg of ice. The organic layer isseparated and the aqueous layer extracted twice with 500 ml ofchloroform. The combined organic extracts are washed once with 500 ml ofwater, dried over anhydrous sodium sulfate and concentrated in vacuo.Distillation of the residue yields 142 g oftrans-1-chloro-dec-1-en-3-one, (A₅, R₁ =C₂ H₅).

b. A solution of 142 g of the product of (a), 140 g of sodium iodide and500 ml of acetone is refluxed under nitrogen for 4 hours. The acetone isthen removed under reduced pressure and the residue dissolved in 500 mlof water. This mixture is extracted twice with 400 ml of ether, theether extracts combined and washed with 5% aqueous sodium thiosulfate,then with saturated sodium chloride and finally dried over anhydroussodium sulfate. The ether is removed in vacuo to yieldtrans-1-iododec-1-en-3-one (A₄, R₁ =C₂ H₅).

c. The crude product of (b) is dissolved in 750 ml of benzene, cooled onan ice bath under nitrogen and then treated with 140 ml of 65% sodiumbis(2-methoxy ethoxy)aluminium hydride over a one hour period. Afterstirring an additional 30 minutes at 0° C, 38 ml of concentratedsulfuric acid in 120 ml of water are added to the reaction mixture. Thereaction mixture is then filtered and the filtrate washed twice with 500ml of saturated sodium chloride. The benzene is removed in vacuo and theresidue distilled to yield 159 g of dl-trans-1-iodo-3-hydroxy-1-decene,(A₃, R₁ =C₂ H₅).

d. A solution of 5.64 g of the product of (c) in 8 ml of isopropenylmethyl ether is cooled to 0° C and treated with 5 drops of ofdichloroacetic acid. The ice bath is then removed and the reactionallowed to proceed at room temperature for 1 hour. Five drops oftriethyl amine are then added and the excess isopropenyl methyl etherremoved in vacuo to yield 7.5 g ofdl-trans-1-iodo-3-(2,2-methoxypropoxy)-1-decene, (A₂, R₁ =C₂ H₅).

e. 7.5 g of the product of (d) are dissolved in 30 ml of ether andcooled to -78° C under nitrogen. 32 ml of 1.25 N t-butyl lithium arethen added over 30 minutes while maintaining the reaction temperaturenear -70° C. The reaction mixture is allowed to stir at -78° C for 45minutes to yield dl-trans-1-lithio-3-(2,2-methoxypropoxy)-1-decene, (A₁,R₁ =C₂ H₅).

f. The thus obtained solution of lithium reagent is added to a solutionof 2.60 g of copper pentyne and 7.9 ml of hexamethyl phosphoroustriamide in 100 ml of ether, also at -78° C. This mixture is allowed tostir at -78° C for 15 minutes to yielddl-1-pentynyl-1-[trans-3-(2,2-methoxypropoxy)-1-decenyl]cuprate, (A, R₁=C₂ H₅).

Similarly, substituting hexanoyl chloride and heptanoyl chloride foroctanoyl chloride in step (a), and by following the procedure asdescribed in steps (a) through (f) above,dl-1-pentynyl-1-[trans-3-(2,2-methoxypropoxy)-1-octenyl]cuprate, (A, R₁=H), anddl-1-pentynyl-1-[trans-3-(2,2-methoxypropoxy)-1-nonenyl]cuprate, (A, R₁=CH₃), are respectively prepared.

PREPARATION 2

This preparation illustrates methods of preparing methyldl-S-keto-15α(β)-(2,2-methoxypropoxy)-20-ethyl-prost-13(t)-enoate (IV,R₁ =C₂ H₅, Z=CH₂ CH₂).

In this preparation a solution of 4.0 g of2-(6-carbomethoxyhexyl)cyclopent-2-en-1-one (B, Z=CH₂ CH₂) in 10 ml ofether is added to a freshly prepared solution ofdl-1-pentynyl-1-[trans-3-(2,2-methoxypropoxy-1-decenyl]cuprate (A, R₁=C₂ H₅), prepared according to Preparation 1. The reaction mixture isstirred at -78° C for 1 hour and then poured into 250 ml of ice water.The organic layer is separated and the aqueous layer extracted twicewith 100 ml of ether. The combined organic layers are dried overanhydrous sodium sulfate and concentrated in vacuo to yield a mixture ofmethyl dl-9-keto-15α-(2,2-methoxypropoxy)-20-ethyl-prost-13(t)-enoateand methyldl-9-keto-15β-(2,2-methoxypropoxy)-20-ethyl-prost-13(t)-enoate.

Similarly, by following the same procedure but replacingdl-1-pentynyl-1-[trans-3-(2,2-methoxypropoxy)-1-decenyl]cuprate bydl-1-pentynyl-1-[trans-3-(2,2-methoxypropoxy)-1-octenyl]cuprate or bydl-1-pentynyl-1-[trans-3-(2,2-methoxypropoxy)-1-nonenyl]cuprate, thefollowing compounds of formula IV:

methyl dl-9-keto-15α-(2,2-methoxypropoxy)-prost-13(t)-enoate and methyldl-9-keto-15β-(2,2-methoxypropoxy)-prost-13(t)-enoate;

methyl dl-9-keto-15α-(2,2-methoxypropoxy)-20 -methyl-prost-13(t)-enoateand methyl dl-9-keto-15β-(2,2-methoxypropoxy)-20-methyl-prost-13(t)-enoate.

In a like manner, substituting2-(6-carbomethoxy-2-cis-hexenyl)-cyclopent-2-en-1-one for2-(6-carbomethoxyhexyl)-cyclopent-2-en-1-one yields a mixture of methyldl-9-keto-15α-(2,2-methoxypropoxy)-20-ethyl-prosta-5(c), 13(t)-dienoateand methyldl-9-keto-15β-(2,2-methoxypropoxy)-20-ethyl-prosta-5(c),13(t)-dienoate.

Similarly, substitutingdl-1-pentynyl-1-[trans-3-(2,2-methoxypropoxy)-1-octenyl]cuprate ordl-1-pentynyl-1-[trans-3-(2,2-methoxypropoxy)-1-nonenyl]cuprate andsubstituting 2-(6-carbomethoxy-2-cis-hexenyl)-cyclopent-2-en-1-one for2-(6-carbomethoxyhexyl)-cyclopent-2-en-1-one and following the sameprocedure as described above yields the following compoundsrespectively: methyl dl-9-keto-15α-(2,2-methoxypropoxy)-prosta-5(c),13(t)-dienoate and methyldl-9-keto-15β-(2,2-methoxypropoxy)-prosta-5(c), 13(t)-dienoate; methyldl-9-keto-15α-(2,2-methoxypropoxy)-20-methyl-prosta-5(c), 13(t)-dienoateand methyldl-9-keto-15β-(2,2-methoxypropoxy)-20-methyl-prosta-5(c),13(t)-dienoate.

PREPARATION 3

This preparation illustrates methods for removing the ether protectinggroup (2,2-methoxypropoxy) from the products of formula IV ofPreparation 2. In this preparation, the mixture of methyldl-9-keto-15α-(2,2-methoxypropoxy)-20-ethyl-prost-13(t)-enoate andmethyl dl-9-keto-15β-(2,2-methoxypropoxy)-20-ethyl-prost-13(t)-enoate(IV, R₁ =C₂ H₅, Z=CH₂ CH₂) obtained in Preparation 2 is dissolved in 50ml of water, 50 ml of methanol and 20 ml of acetic acid and stirred atroom temperature for 1 hour. The reaction mixture is diluted with 100 mlof water and extracted three times with 200 ml of ether. The etherlayers are washed with 500 ml of saturated sodium chloride, dried overanhydrous sodium sulfate and concentrated in vacuo. The residue thusobtained is chromatographed on 400 g of silica gel, eluting with 20%ethyl acetate - hexane (v/v), to yield 2.509 g of methyldl-9-keto-15β-hydroxy-20-ethyl-prost-13(t)-enoate (V, 15β-OH, R₁ =C₂ H₅,Z=CH₂ CH₂). Further elution with 25% ethyl acetate - hexane yields 2.7 gof methyl dl-9-keto-15α-hydroxy-20-ethyl-prost-13(t)-enoate (V, 15α-OH,R₁ =C₂ H₅, Z=CH₂ CH₂).

Similarly, by following the same procedure as above, the etherprotecting groups are removed from the remaining ether protectedproducts of Preparation 2 to yield the following compounds of formula Vwhich can be separated into the respective isomers by thin-layerpreparative chromatography as described above:

methyl dl-9-keto-15α-hydroxy-prost-13(t)-enoate;

methyl-dl-9-keto-15β-hydroxy-prost-13(t)-enoate;

methyl dl-9-keto-15α-hydroxy-20-methyl-prost-13(t)-enoate;

methyl dl-9-keto-15β-hydroxy-20-methyl-prost-13(t)-enoate;

methyl dl-9-keto-15α-hydroxy-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9-keto-15β-hydroxy-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9-keto-15α-hydroxy-prosta-5(c),13(t)-dienoate;

methyl dl-9keto-15β-hydroxy-prosta-5(c),13(t)-dienoate;

methyl dl-9-keto-15α-hydroxy-20-methyl-prosta-5(c),13(t)-dienoate;

methyl dl-9-keto-15β-hydroxy-20-methyl-prosta-5(c),13(t)-dienoate.

PREPARATION 4

This preparation illustrates methods for preparing methyldl-9α,15α-dihydroxy-20-ethyl-prost-13(t)-enoate (VI, 15α-OH, 9α-OH, R₁=C₂ H₅, Z=CH₂ CH₂), and methyldl-9β,15α-dihydroxy-20-ethyl-prost-13(t)-enoate (VI, 15α-OH, 9β-OH, R₁=C₂ H₅, Z=CH₂ CH₂).

In this preparation, a solution of 1.7 g of methyldl-9-keto-15α-hydroxy-20-ethyl-prost-13(t)-enoate, prepared according toPreparation 3, in 100 ml of ethanol is cooled on an ice bath and treatedwith 0.50 g of sodium borohydride. After 45 minutes at 0° C the reactionis quenched by addition of 1 ml of acetic acid. The reaction mixture isthen diluted with 100 ml of water and extracted three times with 200 mlof ethyl acetate. The combined ethyl acetate extracts are washed withsaturated aqueous sodium chloride, dried over anhydrous sodium sulfateand concentrated in vacuo. The residue is then chromatographed on 300 gof silica gel. Elution with 25% ethyl acetate/hexane (v/v) yields 425 mgof methyl dl-9α,15α-dihydroxy-20-ethyl-prost-13(t)-enoate. Furtherelution with 35% ethyl acetate/hexane yields 1.12 g of methyldl-9β,15α-dihydroxy-20-ethyl-prost-13(t)-enoate.

In a like manner, substituting the other methyl ester 9-keto-compoundsprepared in Preparation 3, i.e.,

methyl dl-9-keto-15β-hydroxy-20-ethyl-prost-13(t)-enoate;

methyl dl-9-keto-15α-hydroxy-prost-13(t)-enoate;

methyl dl-9-keto-15β-hydroxy-prost-13(t)-enoate;

methyl dl-9-keto-15α-hydroxy-20-methyl-prost-13(t)-enoate;

methyl dl-9-keto-15β-hydroxy-20-methyl-prost-13(t)-enoate;

methyl dl-9-keto-15α-hydroxy-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9-keto-15β-hydroxy-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9-keto-15α-hydroxy-prosta-5(c),13(t)-dienoate;

methyl dl-9-keto-15β-hydroxy-prosta-5(c),13(t)-dienoate;

methyl dl-9-keto-15α-hydroxy-20-methyl-prosta-5(c),13(t)-dienoate; and

methyl dl-9-keto-15β-hydroxy-20-methyl-prosta-5(c),13(t)-dienoate, for

methyl dl-9-keto-15α-hydroxy-20-ethyl-prost-13(t)-enoate

yields the following compounds of formula VI which are separated intothe respective isomers by thin-layer preparative chromatography:

methyl dl-9α,15β-dihydroxy-20-ethyl-prost-13(t)-enoate, and

methyl dl-9β,15β-dihydroxy-20-ethyl-prost-13(t)-enoate;

methyl dl-9α,15α-dihydroxy-prost-13(t)-enoate, and

methyl dl-9β,15α-dihydroxy-prost-13(t)-enoate;

methyl dl-9α,15β-dihydroxy-prost-13(t)-enoate, and

methyl dl-9β,15β-dihydroxy-prost-13(t)-enoate;

methyl dl-9α,15α-dihydroxy-20-methyl-prost-13(t)-enoate, and

methyl dl-9β,15α-dihydroxy-20-methyl-prost-13(t)-enoate;

methyl dl-9α,15β-dihydroxy-20-methyl-prost-13(t)-enoate, and

methyl dl-9β,15β-dihydroxy-20-methyl-prost-13(t)-enoate;

methyl dl-9α,15α-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoate, and

methyl dl-9β,15α-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9α,15β-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoate and

methyl dl-9β,15β-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9α,15α-dihydroxy-prosta-5(c),13(t)-dienoate, and

methyl dl-9β,15α-dihydroxy-prosta-5(c),13(t)-dienoate;

methyl dl-9α,15β-dihydroxy-prosta-5(c),13(t)-dienoate, and

methyl dl-9β,15β-dihydroxy-prosta-5(c),13(t)-dieonate;

methyl dl-9α,15α-dihydroxy-20 -methyl-prosta-5(c),13(t)-dienoate, and

methyl dl-9β,15α-dihydroxy-20-methyl-prosta-5(c),13(t)-dieonate;

methyl dl-9α,15β-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoate and

methyl dl-9β,15β-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoate.

PREPARATION 5

This preparation illustrates methods for preparing methyldl-9α-hydroxy-15-keto-20-ethyl-prost-13(t)-enoate (VII, 9α-OH, R₁ =C₂H₅, Z=CH₂ CH₂). In this preparation, a solution of 2.006 g of methyldl-9α,15α-dihydroxy-20-ethyl-prost-13(t)-enoate, prepared according toPreparation 4, in 100 ml of benzene is stirred with 3.5 g ofdichlorodicyano quinone for 36 hours at room temperature. The reactionmixture is then diluted with 100 ml of benzene, washed with 100 ml of 5%aqueous sodium bisulfite, 200 ml of saturated aqueous sodium bicarbonateand dried over anhydrous sodium sulfate. The benzene solution isconcentrated in vacuo and the residue chromatographed on 300 g of silicagel. Elution with 20% ethyl acetatehexane (v/v) yields 1.188 g of methyldl-9α-hydroxy-15-keto-20-ethyl-prost-13(t)-enoate.

In a like manner, substituting methyldl-9α,15β-dihydroxy-20-ethyl-prost-13(t)-enoate for methyldl-9α,15α-dihydroxy-20-ethyl-prost-13(t)-enoate yields methyldl-9α-hydroxy-15-keto-20-ethyl-prost-13(t)-enoate.

Likewise, substituting

methyl dl-9β,15α-dihydroxy-20-ethyl-prost-13(t)-enoate or

methyl dl-9β,15β-dihydroxy-20-ethyl-prost-13(t)-enoate;

methyl dl-9α,15α-dihydroxy-prost-13(t)-enoate or

methyl dl-9α,15β-dihydroxy-prost-13(t)-enoate;

methyl dl-9β,15α-dihydroxy-prost-13(t)-enoate or

methyl dl-9β,15βdihydroxy-prost-13(t)-enoate;

methyl dl-9α,15α-dihydroxy-20-methyl-prost-13(t)-enoate or

methyl dl-9α,15β-dihydroxy-20-methyl-prost-13(t)-enoate;

methyl dl-9β,15α-dihydroxy-20-methyl-prost-13(t)-enoate or

methyl dl-9β,15β-dihydroxy-20-methyl-prost-13(t)-enoate,

methyl dl-9α,15α-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoate or

methyl dl-9α,15β-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9β,15α-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoate or

methyl dl-9β,15β-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9α,15α-dihydroxy-prosta-5(c),13(t)-dienoate or

methyl dl-9α,15β-dihydroxy-prosta-5(c),13(t)-dienoate;

methyl dl-9β,15α-dihydroxy-prosta-5(c),13(t)-dienoate or

methyl dl-9β,15β-dihydroxy-prosta-5(c),13(t)-dienoate;

methyl dl-9α,15α-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoate or

methyl dl-9α,15β-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoate;

methyl dl-9β,15α-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoate or

methyl dl-9β,15β-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoate;

respectively, for methyl dl-9α,15α-dihydroxy-20-ethyl-prost-13(t)-enoateas starting materials yields the following compounds of formula VII:

methyl dl-9β-hydroxy-15-keto-20-ethyl-prost-13(t)-enoate;

methyl dl-9α-hydroxy-15-keto-prost-13(t)-enoate;

methyl dl-9β-hydroxy-15-keto-prost-13(t)-enoate;

methyl dl-9α-hydroxy-15-keto-20-methyl-prost-13(t)-enoate;

methyl dl-9β-hydroxy-15-keto-20-methyl-prost-13(t)-enoate;

methyl dl-9α-hydroxy-15-keto-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9β-hydroxy-15-keto-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9α-hydroxy-15-keto-prosta-5(c),13(t)-dienoate;

methyl dl-9β-hydroxy-15-keto-prosta-5(c),13(t)-dienoate;

methyl dl-9α-hydroxy-15-keto-20-methyl-prosta-5(c),13(t)-dienoate; and

methyl dl-9β-hydroxy-15-keto-20-methyl-prosta-5(c),13(t)-dienoate;respectively.

PREPARATION 6

This preparation illustrates methods for preparing methyl dl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoate (VIII, 9α-OH, 15α-OH,15β-CH₃, R₁ =C₂ H₅, Z=CH₂ CH₂), and its isomer methyl dl-9α,15β-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoate, (VIII, 9α-OH, 15β-OH,15α-CH₃, R₁ =C₂ H₅, Z=CH₂ CH₂).

In this preparation, a solution of 1.188 g of methyldl-9α-hydroxy-15-keto-20-ethyl-prost-13(t)-enoate, prepared according toPreparation 5, in 70 ml of tetrahydrofuran is cooled to -30° C andtreated with 6.0 ml of 3 N methyl magnesium bromide in tetrahydrofuran.After stirring for 45 minutes at -30° C, the reaction is quenched by theaddition of 3 ml of acetone and then poured into 200 ml of ice water.The aqueous solution is then extracted three times with 65 ml of ethylacetate and the combined ethyl acetate extracts washed with 300 ml ofsaturated aqueous sodium chloride. The organic layer is then dried overanhydrous sodium sulfate and concentrated in vacuo. The thus-obtainedresidue is chromatographed on 350 g of silica gel. Elution with 20%ethyl acetate - hexane (v/v) yields 0.511 g of methyldl-9α,15β-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoate. Furtherelution with 25% ethyl acetate - hexane (v/v) yields 0.454 g of methyldl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoate.

In a like manner, substituting

methyl dl-9β-hydroxy-15-keto-20-ethyl-prost-13(t)-enoate;

methyl dl-9α-hydroxy-15-keto-prost-13(t)-enoate;

methyl dl-9β-hydroxy-15-keto-prost-13(t)-enoate;

methyl dl-9α-hydroxy-15-keto-20-methyl-prost-13(t)-enoate;

methyl dl-9β-hydroxy-15-keto-20-methyl-prost-13(t)-enoate;

methyl dl-9α-hydroxy-15-keto-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9β-hydroxy-15-keto-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9α-hydroxy-15-keto-prosta-5(c),13(t)-dienoate;

methyl dl-9β-hydroxy-15-keto-prosta-5(c),13(t)-dienoate;

methyl dl-9α-hydroxy-15-keto-20-methyl-prosta-5(c),13(t)-dienoate; and

methyl dl-9β-hydroxy-15-keto-20-methyl-prosta-5(c),13(t)-dienoate

for methyl dl-9α-hydroxy-15-keto-20-ethyl-prost-13(t)-enoate

and following the procedure as described above, yields the followingpair of compounds of formula VIII respectively, which are separated bythin-layer chromatography:

methyl dl-9β,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoate and

methyl dl-9β,15β-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoate

methyl dl-9α,15α-dihydroxy-15β-methyl-prost-13(t)-enoate and

methyl dl-9α,15β-dihydroxy-15α-methyl-prost-13(t)-enoate;

methyl dl-9β,15α-dihydroxy-15β-methyl-prost-13(t)-enoate and

methyl dl-9β,15β-dihydroxy-15α-methyl-prost-13(t)-enoate;

methyl dl-9α,15α-dihydroxy-15β-methyl-20-methyl-prost-13(t)-enoate and

methyl dl-9α,15β-dihydroxy-15α-methyl-20-methyl-prost-13(t)-enoate;

methyl dl-9β,15α-dihydroxy-15β-methyl-20-methyl-prost-13(t)-enoate and

methyl dl-9β,15β-dihydroxy-15α-methyl-20-methyl-prost-13(t)-enoate;

methyldl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prosta-5(c),13(t)-dienoate and

methyldl-9α,15β-dihydroxy-15α-methyl-20-ethyl-prosta-5(c),13(t)-dienoate;

methyldl-9β,15α-dihydroxy-15β-methyl-20-ethyl-prosta-5(c),13(t)-dienoate and

methyldl-9β,15β-dihydroxy-15α-methyl-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9α,15α-dihydroxy-15β-methyl-prosta-5(c),13(t)-dienoate and

methyl dl-9α,15β-dihydroxy-15α-methyl-prosta-5(c),13(t)-dienoate;

methyl dl-9β,15α-dihydroxy-15β-methyl-prosta-5(c),13(t)-dienoate and

methyl dl-9β,15β-dihydroxy-15α-methyl-prosta-5(c),13(t)-dienoate;

methyldl-9α,15α-dihydroxy-15β-methyl-20-methyl-prosta-5(c),13(t)-dienoate and

methyldl-9α,15β-dihydroxy-15α-methyl-20-methyl-prosta-5(c),13(t)-dienoate;

methyldl-9β,15α-dihydroxy-15β-methyl-20-methyl-prosta-5(c),13(t)-dienoate and

methyldl-9β,15β-dihydroxy-15α-methyl-20-methyl-prosta-5(c),13(t)-dienoate.

PREPARATION 7

This preparation illustrates methods for preparing methyldl-9-keto-15α-hydroxy-15β-methyl-20-ethyl-prost-13(t)-enoate (IX,15α-OH, 15β-CH₃, R₁ =C₂ H₅, Z=CH₂ CH₂).

In this preparation, a suspension of 1.00 g of Celite (diatomaceousearth), 1.60 g of chromium trioxide and 53 ml of anhydrous methylenechloride is stirred under nitrogen while 2.29 g of pyridine are added.The resulting suspension is stirred at room temperature for 30 minutes.A solution of 0.94 g of methyldl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoate preparedaccording to Preparation 6, in 5 ml of methylene chloride is added.After 30 minutes at room temperature, the reaction mixture is filteredthrough 50 g of alumina. The alumina is washed several times withmethylene chloride and the combined filtrates concentrated under reducedpressure to yield 0.76 g of methyldl-9-keto-15α-hydroxy-15β-methyl-20-ethyl-prost-13(t)-enoate.

Similarly, substituting methyldl-9β,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoate for methyldl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoate yields methyldl-9-keto-15α-hydroxy- 15β-methyl-20-ethyl-prost-13(t)-enoate.

In a like manner, substituting

methyl dl-9α,15β-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoate or

methyl dl-9β,15β-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoate;

methyl dl-9α,15β-dihydroxy-15α-methyl-prost-13(t)-enoate or

methyl dl-9β,15β-dihydroxy-15α-methyl-prost-13(t)-enoate;

methyl dl-9α,15α-dihydroxy-15β-methyl-prost-13(t)-enoate or

methyl dl-9β,15α-dihydroxy-15β-methyl-prost-13(t)-enoate;

methyl dl-9α,15β-dihydroxy-15α-methyl-20-methyl-prost-13(t)-enoate or

methyl dl-9β,15β-dihydroxy-15α-methyl-20-methyl-prost-13(t)-enoate;

methyl dl-9α,15α-dihydroxy-15β-methyl-20-methyl-prost-13(t)-enoate or

methyl dl-9β,15α-dihydroxy-15β-methyl-20-methyl-prost-13(t)-enoate;

methyldl-9α,15β-dihydroxy-15α-methyl-20-ethyl-prosta-5(c),13(t)-dienoate or

methyldl-9β,15β-dihydroxy-15α-methyl-20-ethyl-prosta-5(c),13(t)-dienoate;

methyldl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prosta-5(c),13(t)-dienoate or

methyldl-9β,15α-dihydroxy-15β-methyl-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9α,15β-dihydroxy-15α-methyl-prosta-5(c),13(t)-dienoate or

methyl dl-9β,15β-dihydroxy-15α-methyl-prosta-5(c),13(t)-dienoate;

methyl dl-9α,15α-dihydroxy-15β-methyl-prosta-5(c),13(t)-dienoate or

methyl dl-9β,15α-dihydroxy-15β-methyl-prosta-5(c),13(t)-dienoate;

methyldl-9α,15β-dihydroxy-15α-methyl-20-methyl-prosta-5(c),13(t)-dienoate or

methyldl-9β,15β-dihydroxy-15α-methyl-20-methyl-prosta-5(c),13(t)-dienoate.

methyl dl-9α,15α-dihydroxy-15β-methyl-20-methyl-prosta-5(c),13(t)-dienoate or

methyldl-9β,15α-dihydroxy-15β-methyl-20-methyl-prosta-5(c),13(t)-dienoate;

respectively for methyldl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoate as startingmaterial and following the procedure described above yields thecompounds of formula IX listed herebelow:

methyl dl-9-keto-15β-hydroxy-15α-methyl-20-ethyl-prost-13(t)-enoate;

methyl dl-9-keto-15β-hydroxy-15α-methyl-prost-13(t)-enoate;

methyl dl-9-keto-15α-hydroxy-15β-methyl-prost-13(t)-enoate;

methyl dl-9-keto-15β-hydroxy-15α-methyl-20-methyl-prost-13(t)-enoate;

methyl dl-9-keto-15α-hydroxy-15β-methyl-20-methyl-prost-13(t)-enoate;

methyldl-9-keto-15β-hydroxy-15α-methyl-20-ethyl-prosta-5(c),13(t)-dienoate;

methyldl-9-keto-15α-hydroxy-15β-methyl-20-ethyl-prosta-5(c),13(t)-dienoate;

methyl dl-9-keto-15β-hydroxy-15α-methyl-prosta-5(c),13(t)-dienoate;

methyl dl-9-keto-15α-hydroxy-15β-methyl-prosta-5(c),13(t)-dienoate;

methyldl-9-keto-15β-hydroxy-15α-methyl-20-methyl-prosta-5(c),13(t)-dienoate;and

methyldl-9-keto-15α-hydroxy-15β-methyl-20-methyl-prosta-5(c),13(t)-dienoate;

respectively.

PREPARATION 8

This preparation illustrates methods for preparingdl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acid, (VIII,9α-OH, 15α-OH, 15β-CH₃, free acid, R₁ =C₂ H₅, Z=CH₂ CH₂).

In this preparation, a solution of 0.454 g of methyldl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoate, preparedaccording to Preparation 6, 0.75 g of potassium hydroxide, 10 ml ofmethanol and 10 ml of water is stirred at room temperature undernitrogen for 1 hour and 45 minutes. The reaction mixture is diluted with50 ml of water and washed with 100 ml of ether. The aqueous layer isthen acified to pH 4 with 1 N hydrochloric acid, saturated with sodiumchloride, and extracted three times with 75 ml of ethyl acetate. Thecombined ethyl acetate extracts are washed with 300 ml of saturatedaqueous sodium chloride and dried over anhydrous sodium sulfate.Concentration of the organic solution gives a residue which isrecrystallized from 1 ml of ethyl acetate and 10 ml of hexane. Oncooling overnight at -20° C, 0.329 g ofdl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acidprecipitates and is collected by filtration.

Similarly, substituting the other compounds obtained in Preparation 6for methyl dl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoateand following the procedure as described above yields the following freeacids, corresponding to compounds of formula VIII:

di-9α,15β-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid;

dl-9β,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acid;

dl-9β,15β-dihydroxy-15α-methyl-29-ethyl-prost-13(t)-enoic acid;

dl-9α,15α-dihydroxy-15β-methyl-prost-13(t)-enoic acid;

dl-9α,15β-dihydroxy-15α-methyl-prost-13(t)-enoic acid;

dl-9β,15α-dihydroxy-15β-methyl-prost-13(t)-enoic acid;

dl-9β ,15β-dihydroxy-15α-methyl-prost-13(t)-enoic acid;

dl-9α,15α-dihydroxy-15β-methyl-20-methyl-prost-13(t)-enoic acid;

dl-9α,15β-dihydroxy-15α-methyl-20-methyl-prost-13(t)-enoic acid;

dl-9β,15α-dihydroxy-15β-methyl-20-methyl-prost-13(t)-enoic acid;

dl-9β,15β-dihydroxy-15α-methyl-20-methyl-prost-13(t)-enoic acid;

dl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15β-dihydroxy-15α-methyl-29-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15α-dihydroxy-15β-methyl-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15β-dihydroxy-15α-methyl-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15α-dihydroxy-15β-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15β-dihydroxy-15α-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15α-dihydroxy-15β-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15β-dihydroxy-15α-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15α-dihydroxy-15β-methyl-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15β-dihydroxy-15α-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15α-dihydroxy-15β-methyl-20-methyl-prosta-5(c),13(t)-dienoic acid;and

dl-9β,15β-dihydroxy-15α-methyl-20-methyl-prosta-5(c),13(t)-dienoic acid.

In a like manner, substituting the compounds prepared in Preparation 7for methyl-dl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoateand following the procedure as described above yields the following freeacids, corresponding to compounds of formula IX:

dl- 9-keto-15α-hydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acid;

dl-9-keto-15β-hydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid;

dl-9-keto-15α-hydroxy-15β-methyl-prost-13(t)-enoic acid;

dl-9-keto-15β-hydroxy-15α-methyl-prost-13(t)-enoic acid;

dl-9-keto-15α-hydroxy-15β-methyl-20-methyl-prost-13(t)-enoic acid;

dl-9-keto-15β-hydroxy-15α-methyl-20-methyl-prost-13(t)-enoic acid;

dl-9-keto-15α-hydroxy-15β-methyl-20-ethyl-prosta-5(c),13(t)-dienoicacid;

dl-9-keto-15β-hydroxy-15α-methyl-20-ethyl-prosta-5(c),13(t)-dienoicacid;

dl-9-keto-15α-hydroxy-15β-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15β-hydroxy-15α-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15α-hydroxy-15β-methyl-20-methyl-prosta-5(c),13(t)-dienoicacid; and

dl-9-keto-15β-hydroxy-15α-methyl-20-prosta-5(c),13(t)-dienoic acid.

Also, substituting the compounds prepred in Preparation 4 for methyldl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoate and followingthe procedure as described above yields the following free acids,corresponding to compounds of formula VI:

dl-9α,15α-dihydroxy-20-ethyl-prost-13(t)-enoic acid;

dl-9β,15α-dihydroxy-20-ethyl-prost-13(t)-enoic acid;

dl-9α,15β-dihydroxy-20-ethyl-prost-13(t)-enoic acid;

dl-9β,15β-dihydroxy-20-ethyl-prost-13(t)-enoic acid;

dl-9α,15α-dihydroxy-prost-13(t)-enoic acid;

dl-9β,15α-dihydroxy-prost-13(t)-enoic acid;

dl-9α,15β-dihydroxy-prost-13(t)-enoic acid;

dl-9β,15β-dihydroxy-prost-13(t)-enoic acid;

dl-9α,15α-dihydroxy-20-methyl-prost-13(t)-enoic acid;

dl-9β,15α-dihydroxy-20-methyl-prost-13(t)-enoic acid;

dl-9α,15β-dihydroxy-20-methyl-prost-13(t)-enoic acid;

dl-9β,15β-dihydroxy-20-methyl-prost-13(t)-enoic acid;

dl-9α,15α-dihydroxy-20-ethyl-prost-5(c),13(t)-dienoic acid;

dl-9β,15α-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15β-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15β-dihydroxy-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15α-dihydroxy-prosta-5(c),13(t)-dienoic acid;

dl-9β,15α-dihydroxy-prosta-5(c),13(t)-dienoic acid;

dl-9α,15β-dihydroxy-prosta-5(c),13(t)-dienoic acid;

dl-9β,15β-dihydroxy-prosta-5(c),13(t)-dienoic acid;

dl-9α,15α-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9β,15α-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoic acid;

dl-9α,15β-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoic acid; and

dl-9β,15β-dihydroxy-20-methyl-prosta-5(c),13(t)-dienoic acid.

Similarly, substituting the compounds prepared in Preparation 3 formethyl dl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoate andfollowing the procedure as described above yields the following freeacids, corresponding to compounds of formula V:

dl-9-keto-15α-hydroxy-20-ethyl-prost-13(t)-enoic acid;

dl-9-keto-15β-hydroxy-20-ethyl-prost-13(t)-enoic acid;

dl-9-keto-15α-hydroxy-prost-13(t)-enoic acid;

dl-9-keto-15β-hydroxy-prost-13(t)-enoic acid;

dl-9-keto-15α-hydroxy-20-methyl-prost-13(t)-enoic acid;

dl-9-keto-15β-hydroxy-20-methyl-prost-13(t)-enoic acid;

dl-9-keto-15α-hydroxy-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15β-hydroxy-20-ethyl-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15α-hydroxy-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15β-hydroxy-prosta-5(c),13(t)-dienoic acid;

dl-9-keto-15α-hydroxy-20-methyl-prosta-5(c),13(t)-dienoic acid and

dl-9-keto-15β-hydroxy-20-methyl-prosta-5(c),13(t)-dienoic acid.

In this Application use is made of the microbiological Classificationaccording to the scheme proposed by Ainsworth (1966):

"A general purpose classification of fungi -- Bibliography of SystematicMycology (1966), 1-4 -- Commonwealth Mycological Institute -- Kew,Surrey", and use is made of Ainsworth and Bibsy's Dictionary of theFungi, 6th edition (1971).

The aforesaid Division of Eumycota embraces 5 Sub-divisions, viz.Mastigomycotina, Deuteromycotina, Basidiomycotina, Ascomycotina andZygomycotina. While numerous species of microorganisms falling withinthe 5 Sub-divisions of Eumycota can be employed in the process of theinvention for the preparation of the 18 , 19 - and 20-hydroxy-prostaglandin derivatives of formula I supra, it is preferredto employ species of microorganisms falling within the Classes andOrders listed hereinbelow:

Mastigomycotina

Oomycetes

Saprolegniales

Peronosporales

Deuteromycotina

Coelomycetes

Sphaeropsidales

Melanconiales

Hyphomycetes

Hyphomycetales

Tuberculariales

Basidiomycotina

Gasteromycetes

Lycoperdales

Hymenomycetes

Aphyllophorales

Agaricales

Ascomycotina

Plectomycetes

Eurotiales

Microascales

Pyrenomycetes

Sphaeriales

Hypocreales

Loculoascomycetes

Pleosporales

Zygomycotina

Zygomycetes

Mucorales

Entomophthorales

While numerous species of microorganisms falling within the Family ofStreptomycetaceae can be employed in the process of the invention forthe preparation of the 18 - and 19 -hydroxy-prostaglandin derivatives ofgeneral formula I, it is preferred to employ species of microorganismsfalling within the genus Streptomyces.

Cultures of a large number of species, falling within the group ofmicroorganisms which can be employed in the process of the invention,are available from known sources, such as:

"Centraal Bureau voor Schimmelcultures" (CBS), Baarn, The Neatherlands;

"American Type Culture Collection" (ATCC), Rockville, Maryland, U.S.A.;

"northern Utilization Research and Development Division of U.S.Department of Agriculture" (NRRL), Peoria, Illinois, U.S.A. and

"Commonwealth Mycological Institute" (CMI), Kew, Surrey, England.

The microorganisms to be used is grown in the conventional way,preferably in a liquid medium with constant aeration by shaking or bystirring while passing through air. Culture media used for the growth offungal organisms and Streptomyces are well known in the art andprincipally consist of (1) a source of carbon such as glucose, maltose,sucrose, starch, dextrine and vegetable oils and (2) a source ofnitrogen such as ammonia salts, meat and fish flours, corn steep solidsand other nutritive substances containing nitrogen, (3) inorganic saltssuch as sodium, potassium, magnesium, sulphates, phosphates andchlorides, and, optionally, trace elements. The foregoing materials areadded in the desired amounts to a quantity of tap water, and thesolution is sterilized prior to inoculation with the microorganismculture.

The prostaglandin or prostaglandin derivative of general formula II tobe hydroxylated is added in the form of a fine crystal suspension ordissolved in a solvent such as acetone, ethanol or dimethyl formamide.During the incubation of the starting prostaglandin with the fungus orstreptomycete cultures, aeration is provided by shaking and thetemperature is kept between 20° and 40° C during 12-48 hours. Thehydroxylation is followed with the aid of thin-layer chromatography. Thehydroxylated products are isolated from the fermentation broth by knownprocedures. At the end of the fermentation the broth is filtered, thefiltrate acidified to about ph=3 and extracted with a suitable organicsolvent. For acidification either organic or mineral acids can be used,such as phosphoric acid, sulphuric acid, formic acid, and citric acid.Extraction can be carried out at pH between 1 and 5. However, it isadvisable not to work at pH lower than 2, as many prostaglandinderivatives are acid sensitive. Suitable solvents for extraction areketones, esters and ethers, such as methyl isobutyl ketone, ethylacetate and diethyl ether. It is also possible to acidify the culturebroth and extract directly without filtration.

The crude products are purified by known procedures such as directcrystallisation or column chromatography. A suitable adsorbent is forexample silica gel. The silica is normally pre-treated with 20% of watercontaining 1% of acetic acid and the column eluted with suitable organicsolvents or mixtures thereof, such as ethyl acetate - heptane (8.3 v/v)containing 0.1% of acetic acid.

The analysis of the products thus obtained sometimes presents somedifficulty. Mass spectrometry of prostaglandins often yields complexspectra, which are difficult to interpret. Sometimes even the molecularpeak cannot be determined.

Better results are obtained by protecting reactive groups such ashydroxyl groups, keto groups and carboxylic groups by the followingreactions, respectively:

1. esterification of the carboxylic groups by diazomethane;

2. transformation of keto groups into methoximes; and

3. conversion of hydroxyl groups into trimethylsilyloxy groups, forexample with N,O-bis(trimethylsilyl) trifluoro-acetamide.

Such converted products are hereinafter referred to as "protectedproducts". The crude derivative is then injected into a GLC-columnconnected to a double focussing mass spectrometer and the spectrum ofthe largest GLC-peak is recorded. GLC is used to obtain a separation ofmain products from byproducts and to record C-values according to themethod of S. Bergstrom et al., J. Biol. Chem.-238 (1963), 3555.

For the determination of these values mixtures of normal-fatty acids areused as standards. The retention times of the standards are plotted on alogarithmic scale against the number of carbon atoms of the acids on alinear scale. These diagrams are then used to convert observed retentiontimes to C-values.

These C-values are obtained using the following gaschromatographicconditions:

Column: 5 ft, 2.3 mm i.d.

Stationary phase: 3% OV-17 on Gaschrom Q 100-120 mesh

Oven temperature: 235° C

Carrier gas: 38 ml N₂ /min.

The 18 -hydroxy and 19 -hydroxy-prostaglandin derivatives are usuallyobtained as a mixture; the isomers can be separated from each other andeach of the isomers isolated according to the procedures describedhereinabove. Sometimes also 17 -hydroxylated products are obtained asbyproducts. These 17 -hydroxy-prostaglandin derivatives are also novelcompounds. The hydroxylation of PGA₂ is usually preceded by reduction ofthe 10(11) double bond.

The alkyl esters are obtained by treatment of the compounds of generalformula I with an excess of a diazoalkane such as diazomethane,diazoethane or diazopropane in diethyl ether or methylene chloridesolution, in a conventional manner.

Alternatively, the mixture of 18 - and 19 -hydroxylated compounds can beesterified as described immediately above, and the 18 -hydroxy and 19-hydroxy-alkyl esters recovered, purified and/or separated, according toprocedures described above for the compounds of formula I.

The salt derivatives of the acids of formula I are prepared by treatingthe corresponding free acids with about one molar equivalent of asuitable base, such as sodium hydroxide, potassium hydroxide, ammoniumhydroxide, calcium hydroxide, trimethylamine, triethylamine,tripropylamine, β-(dimethylamino) ethanol, β-(diethylamino) ethanol,triethanolamine, arginine, lysine, caffeine, procaine and the like. Thereaction is usually conducted in an aqueous solution, alone or incombination with an inert, water-miscible organic solvent, at atemperature of from about 0° C to about 30° C, preferably at roomtemperature. Typical inert, water-miscible organic solvents includemethanol, ethanol, isopropanol, butanol, dioxane or tetrahydrofuran.When divalent metal salts are prepared, such as the calcium salts ormagnesium salts, the free acid starting material is treated with atleast one half molar equivalent of the base.

The free acids, ester or salts of the 18 -, 19 - and 20-hydroxy-prostaglandin derivatives of general formula I can beadministered in a wide variety of dosage forms, either alone or incombination with other pharmaceutical compatible medicaments, in theform of pharmaceutical compositions suited for oral or parenteraladministration of inhalation. The conpounds are typically administeredas pharmaceutical compositions consisting essentially of the free acids,esters or salts of the invention, and a pharmaceutical carrier. Thepharmaceutical carrier can be either a solid material, liquid oraerosol, in which the free acid, ester or salt is dissolved, dispersedor suspended, and can optionally contain small amounts of preservativesand/or pH-buffering agents. Suitable preservatives which can be usedinclude, for example, benzyl alcohol and the like. Suitable bufferingagents include, for example, sodium acetate and pharmaceutical phosphatesalts and the like.

The liquid compositions can, for example, be in the form of solutions,emulsions, suspensions, syrups, or elixirs. The solid compositions cantake the form of tablets, powders, capsules, pills or the like,preferably in unit dosage forms for simple administration or precisedosages. Suitable solid carriers include, for example, pharmaceuticalgrades of starch, lactose, sodium saccharin, talcum, sodium bisulfiteand the like.

For inhalation administration, the free acids, esters or salts can, forexample, be administered as an aerosol in an inert propellant togetherwith a cosolvent, e.g., ethanol, together with optional preservatives,surfactants, stabilizers, isotonic and buffering agents. Additionalgeneral information concerning the inhalation administration of aerosolscan be had by reference to U.S. Pat. Nos. 2,868,691 and 3,095,355.

For the preparation of an aerosol the active compound is firstmicronized; preferred particle size is from 0.5 to 10 μ. The solutionsor suspensions to be used contain from 0.02 to 0.5 mg of active compoundper ml of pharmaceutically acceptable solvent medium. Preferably, the pHof the solution or suspension is between 4 and 7.

The solutions or suspensions are used in an aerosol container providedwith a metered valve which releases preferably from 50 to 60 μl perpuff. Propellants conventional in pharmaceutical aerosols, such asvarious chloro-fluoro-alkanes, may be used.

A suitable aerosol can be prepared, for example, using solutions orsuspensions and propellants consistingof:__________________________________________________________________________9-keto-11α,15α,19-trihydroxy-prost-13(t)-enoic acidtriethanolamine salt 0.25%ethanolabsolute36.75%dichlorodifluoromethane/1,2-dichloro-1,1,2,2,-tetrafluoroethane(40/60)ad 100%or9-keto-11α,15α ,18-trihydroxy-prost-13(t)-enoic 0.5gpropylene glycol 1 gethanol absolute 19.5gdichlorodifluoromethane/1,2-dichloro-1,1,2,2-tetrafluoroethane (40/60)100g__________________________________________________________________________

The free acids, esters or salts of the invention are typicallyadministered i.v. in dosages of about 0.1 to 10 mg and p.o. in dosagesof about 1 to 100 mg. The daily doses are i.v. about 0.4 to 40 mg andp.o. about 6 to 600 mg.

The following Examples illustrate the invention.

EXAMPLE I

a. An agar slant of Thozetellopsis tocklaiensis (CBS 378.58) was used toinoculate 100 ml of sterile 20-20 medium in a 500 ml conical flask. Thismedium was prepared by solving 20 g of glucose in 500 ml of tap water,adding 20 g of corn steep solids and filling up to 1 liter with tapwater; pH was adjusted to 6.5 with the aid of a 30% solution of sodiumhydroxide. Sterilization was effected during 20 minutes at 120° C.

The flask was incubated during 72 hours at 26° C on a rotary shaker (280r.p.m., 2.5 cm stroke). From the culture obtained 5 ml were used toinoculate 10 ml of sterile 10-10 medium in a 500 ml conical flask. Themedium was prepared as the 20-20 medium described above using 10 g ofglucose and corn steep solids each per liter. The flask was incubated at26° C on the rotary shaker.

18 Hours after inoculation 20 mg ofdl-9-keto-15αhydroxy-prost-13(t)-enoic acid, prepared according toPreparations 3 and 8, dissolved in 2.5 ml of 50% aqueous ethanol, wereadded and the incubation was continued for another 24 hours at 26° C.Hereafter the culture broth was filtered, the filtrate acidified to pH=3with a 10% aqueous citric acid solution, and extracted three times with20 ml of ethyl acetate. The extract was evaporated in vacuo and theresidue purified by column chromatography (SiO₂ pretreated with 1%acetic acid; eluted with ethyl acetate - heptane (8:3) containing 0.1%acetic acid). The matching fractions were combined and evaporated invacuo yielding 2.5 mg of 9-keto-15α, 18 -dihydroxy-prost-13(t)-enoicacid and 3.5 mg of 9-keto-15α, 19 -dihydroxy-prost-13(t)-enoic acid.

The protected 18-hydroxy product (silyl ether, methoxime, methyl ester)has:

C-value: 25.9

Molecular peak in mass spectrum: m/e = 541

Intense peaks: 510, 420, 382, 309, 197, 131, 129.

Minor characteristic fragments: 422, 390, 364, 222, 144.

The protected 19-hydroxy product has:

C-value: 26.2

Molecular peak in mass spectrum: m/e = 541

Intense peaks: 510, 420, 382, 129, 117.

Minor characteristic fragments: 466, 368, 330, 309, 222, 143.

b. In the same way dl-9-keto-15β-hydroxy-prost-13(t)-enoic acid,prepared according to Preparations 3 and 8, was converted into9-keto-15β, 18 -dihydroxy-prost-13(t)-enoic acid and 9-keto-15β, 19-dihydroxy-prost-13(t)-enoic acid.

The protected 18-hydroxy product has:

C-value: 26.0

Molecular peak in mass spectrum: m/e = 541

Intense peaks: 510, 420, 382, 309, 197, 131, 129.

Minor characteristic fragments: 422, 390, 364, 222, 144.

The protected 19-hydroxy product has:

C-value: 26.3

Molecular peak in mass spectrum: m/e = 541

Intense peaks: 510, 420, 382, 129, 117.

Minor characteristic fragments: 466, 368, 330, 309, 222, 143.

c. In the same way dl-9α, 15β-dihydroxy-prost-13(t)-enoic acid, preparedaccording to Preparations 4 and 8, was converted into 9α,15β, 18-trihydroxy-prost-13(t)-enoic acid and 9α, 15β19-trihydroxy-prost-13(t)-enoic acid. The silylated methyl ester of the18-hydroxy compound has:

C-value: 24.3

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 297, 197, 131, 129.

Minor characteristic fragments: 557, 496, 467, 377, 350, 310, 247, 144.

The silylated methyl ester of the 19-hydroxy compound has:

C-value: 24.6

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 297, 197, 143, 129, 117.

Minor characteristic fragments: 496, 452, 310, 247, 143.

d. In the same way dl-9β,15β-dihydroxy-prost-13(t)-enoic acid, preparedaccording to Preparations 4 and 8, was converted into 9β, 15β, 18-trihydroxy-prost-13(t)-enoic acid and 9β, 15β, 19-trihydroxy-prost-13(t)-enoic acid. The silylated methyl ester of the18-hydroxy compound has:

C-value: 24.3

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 247, 197, 131, 129.

Minor characteristic fragments: 557, 467, 377, 350, 297, 223.

The silylated methyl ester of the 19-hydroxy compound has:

C-value: 24.6

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 247, 197, 129, 117.

Minor characteristic fragments: 452, 297, 223.

e. In the same way 9-keto-15α-hydroxy-prosta-5(c),8(12),13(t)-trienoicacid (PGB₂) was converted into 9-keto-15α, 18-dihydroxy-prost-5(c),8(12), 13(t)-trienoic acid and 9-keto-15α,19-dihydroxy-prosta-5(c),8(12),13(t)-trienoic acid. The protected18-hydroxy compound (silyl ether, methyl ester, methoxime) has:

C-value: 27.2

Molecular peak in mass spectrum: m/e = 537

Intense peaks: 506, 416, 360, 131.

Minor characteristic fragments: 418, 378, 326, 162.

The protected 19-hydroxy derivative has:

C-value: 27.7

Molecular peak in mass spectrum: m/e = 537

Intense peaks: 506, 416, 129, 117.

Minor characteristic fragments: 378, 346, 326, 162.

f. In the same way 9-keto-15α-hydroxy-prosta-5(c),10,13(t)-trienoic acid(PGA₂) was converted into 9-keto-15α,18-dihydroxy-prosta-5(c),13(t)-dienoic acid and 9-keto-15α, 19-dihydroxy-prosta-5(c),13(t)-dienoic acid.

The protected 18-hydroxy compound has:

C-value: 25.9

Molecular peak in the mass spectrum: m/e = 539

Intense peaks: 508, 418, 131, 129.

Minor characteristic fragments: 438, 380, 226, 220, 197.

The protected 19-hydroxy derivative has:

C-value: 26.2

Molecular peak in mass spectrum: m/e = 539.

Intense peaks: 508, 418, 380, 348, 143, 129, 117.

Minor characteristic fragments: 438, 226, 220.

g. In the same way dl-9β, 15α-dihydroxy-prost-13(t)-enoic acid, preparedaccording to Preparations 4 and 8, was converted into 9β, 15α, 18-trihydroxy-prost-13(t)-enoic acid and 9β, 15α, 19-trihydroxy-prost-13(t)-enoic acid. The silylated methyl ester of the18-hydroxy compound has:

C-value: 24.3

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 247, 197, 131, 129.

Minor characteristic fragments: 557, 467, 377, 350, 297, 223.

The silylated methyl ester of the 19-hydroxy compound has:

C-value: 24.6

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 247, 197, 129, 117.

Minor characteristic fragments: 452, 297, 223.

h. In the same way dl-9α, 15α-dihydroxy-prost-13(t)-enoic acid, preparedaccording to Preparations 4 and 8, was converted into 9α, 15α, 18-trihydroxy-prost-13(t)-enoic acid and 9α, 15α, 19-trihydroxy-prost-13(t)-enoic acid.

The silylated methyl ester of the 18-hydroxy product has:

C-value: 24.2

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 297, 197, 131, 129.

Minor characteristic fragments: 557, 496, 467, 377, 350, 310, 247, 144.

The silylated methyl ester of the 19-hydroxy compound has:

C-value: 24.5

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 297, 197, 143, 129, 117.

Minor characteristic fragments: 496, 452, 310, 247, 143.

EXAMPLE II

a. An agar slant of Delacroixia coronata (CBS 647.68) was used toinoculate 100 ml of sterile 20-20 medium in a 500 ml conical flask. Thismedium was prepared as described in Example I a.

The flask was incubated during 72 hours at 26° C on a rotary shaker (280r.p.m., 2.5 cm stroke). From the culture obtained 5 ml were used toinoculate 100 ml of sterile 10-10 medium in a 500 ml conical flask. Themedium was prepared as described in Example I a. The flask was incubatedat 26° C on the rotary shaker.

18 Hours after inoculation 2 mg of dl-9α,15α-dihydroxy-15β-methyl-prost-13(t)-enoic acid, prepared according toPreparations 6 and 8, dissolved in 2.5 ml of 50% aqueous ethanol, wereadded and the incubation was continued for another 24 hours at 26° C.Hereafter the culture broth was filtered, the filtrate acidified to pH=3with a 10% aqueous citric acid solution and extracted three times with20 ml of ethyl acetate. The extract was evaporated in vacuo and theresidue purified by column chromatography (SiO₂ pretreated with 1%acetic acid; eluted with ethyl acetate - heptanol (8:3) containing 0.1%acetic acid). The matching fractions were combined and evaporated invacuo yielding 2.0 mg of 9α, 15α, 18-trihydroxy-15β-methyl-prost-13(t)-enoic acid and 3.8 mg of 9α, 15α, 19-trihydroxy-15β-methyl-prost-13(t)-enoic acid.

The silylated methyl ester of the 18-hydroxy product has:

C-value: 24.2

Molecular peak in mass spectrum: m/e = 600

Intense peaks: 441, 351, 297, 211, 142, 131.

Minor characteristic fragments: 585, 571, 481, 323, 301, 257, 144.

The protected 19-hydroxy product has:

C-value: 24.6

Molecular peak in mass spectrum: m/e = 600

Intense peaks: 441, 351, 297, 143, 117.

Minor characteristic fragments: 585, 323, 301, 211.

b. In the same way dl-9-keto-15β-hydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid, prepared according to Preparations 7 and8, was converted into 9-keto-15β, 18-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid and 9-keto-15β, 19-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid.

The protected 18-hydroxy product (silyl ether, methoxime, methyl ester)has:

C-value: 27.0

Molecular peak in mass spectrum: m/e = 583

Intense peaks: 396, 143.

Minor characteristic fragments: 526, 462, 436, 366, 364, 171, 159.

The protected 19-hydroxy product has:

C-value: 27.4

Molecular peak in mass spectrum: m/e = 583

Intense peaks: 396, 171, 145, 143.

Minor characteristic fragments: 462, 450, 366, 364, 239.

EXAMPLE III

a. An agar slant of Streptomyces sp. (CBS 188.74) was used to inoculate100 ml of the following medium in a 500 ml conical flask: peptone 10g/l, malt paste 15 g/l, NaCl 5 g/l, distilled water; the pH was adjustedto 7.2 with the aid of 30% aqueous potassium hydroxide solution.Sterilization was effected for 20 minutes at 120° C.

The flask was incubated during 72 hours at 26° C on a rotary shaker (280r.p.m., 2.5 cm stroke). From the culture obtained 5 ml were used toinoculate 100 ml of the following medium in a 500 ml conical flask:glucose 10 g/l, corn steep solids 3 g/l, peptone 5 g/l, NaCl 5 g/l, tapwater; the pH was adjusted to 7.2 with the aid of a 30% aqueouspotassium hydroxide solution. Sterilization was effected for 20 minutesat 120° C.

The flask was incubated for 72 hours at 26° C on the rotary shaker.Hereafter 20 mg of 9-keto-11α,15α-dihydroxy-prost-13(t)-enoic acid(PGE₁), dissolved in 2.5 ml of 50% aqueous ethanol, were added and theincubation was continued for another 24 hours. According to thin layerchromatography two compounds were formed which were more polar than thestarting material. The fermentation broth was filtered, the filtrateacidified to pH=3 with a 10% aqueous citric acid solution, and extractedthree times with 30 ml of ethyl acetate. The extract was evaporatedunder reduced pressure and the residue purified by column chromatography(SiO₂ pretreated with 1% acetic acid and 19% water; eluted with ethylacetate containing 0.1% acetic acid). The matching fractions werecombined and evaporated under reduced pressure. The less polar of thetwo transformation products was obtained in 5.0 mg yield as an oil andproved to be 9-keto-11α,15α,18 -trihydroxy-prost-13-(t)-enoic acid,according to combined GLC-mass spectrometry. The protected product has:

C-value: 26.6

Molecular peak in mass spectrum: m/e = 629

Intense peaks: 297, 133, 131, 129.

Minor characteristic fragments: 598, 510, 470, 420, 380, 366, 310, 223,197, 144.

The more polar of the transformation products was also obtained as anoil (yield 4 mg). This compound proved to be 9-keto-11α,15α,19-trihydroxyprost-13(t)-enoic acid, according to combined GLC-massspectrometry.

The protected compound has:

C-value: 27.0

Molecular peak in mass spectrum: m/e = 629

Intense peak: 366, 297, 223, 183, 143, 133, 129, 117.

Minor characteristic fragments: 598, 470, 380, 197.

b. In the same way 9-keto-11α,15α-dihydroxy-prosta-5(c),13(t)-dienoicacid (PGE₂) was transformed into

9-keto-11α,15α,18 -trihydroxy-prosta-5(c),13(t)-dienoic acid and

9-keto-11α,15α,19 -trihydroxy-prosta-5(c),13(t)-dienoic acid.

The protected 18-hydroxy compound has:

C-value: 26.6

Molecular peak in mass spectrum: m/e = 627

Intense peaks: 596, 506, 366, 295, 223, 133, 131, 129.

Minor characteristic fragments: 508, 468, 418, 378, 364, 197, 144.

The protected 19-hydroxy compound has:

C-value: 26.9

Molecular peak in mass spectrum: m/e = 627

Intense peaks: 596, 506, 366, 295, 223, 143, 133, 129, 117.

Minor characteristic fragments: 468, 378, 364, 197.

c. In the same way 9α,11α,15α-trihydroxy-prosta-5(c),13(t)-dienoic acid(PGF₂.sub.α) was transformed into 9α,11α,15α,18-tetrahydroxy-prosta-5(c),13(t)-dienoic acid and 9α,11α,15α,19-tetrahydroxy-prosta-5(c),13(t)-dienoic acid.

The protected 18-hydroxy product has:

C-value: 25.0

Molecular peak in mass spectrum: m/e = 672

Intense peaks: 423, 333, 307, 217, 197, 191, 171, 131, 129.

Minor characteristic fragments: 643, 582, 553, 513, 481, 397.

The protected 19-hydroxy product has:

C-value: 25.3

Molecular peak in mass spectrum: m/e = 672

Intense peaks: 423, 333, 307, 217, 197, 191, 143, 129, 117.

Minor characteristic fragments: 657, 582, 567, 531, 513, 481, 397.

d. In the same way dl-9α,15α,-dihydroxy-20-ethyl-prost-13(t)-enoic acid,prepared according to Preparations 4 and 8, was transformed into9α,15α,18 -trihydroxy-20-ethyl-prost-13(t)-enoic acid and 9α,15α,19-trihydroxy-20-ethyl-prost-13(t)-enoic acid.

The protected 18-hydroxy product has:

C-value: 25.6

Molecular peak in mass spectrum: m/e = 614

Intense peaks: 427, 337, 297, 129.

Minor characteristic fragments: 557, 467, 377, 225, 159.

The protected 19-hydroxy compound has:

C-value: 25.9

Molecular peak in mass spectrum: m/e = 614

Intense peaks: 427, 337, 297, 129.

Minor characteristic fragments: 571, 481, 391, 225, 145.

e. In the same waydl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acid, preparedaccording to Preparations 6 and 8, was transformed into 9α,15α,18-trihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acid and 9α,15α,19-trihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acid.

The protected product has:

C-value: 25.3

Molecular peak in mass spectrum: m/e = 628

Intense peaks: 441, 351, 297, 159, 143.

Minor characteristic fragments: 571, 481, 323, 239.

The protected 19-hydroxy derivative has:

C-value: 25.9

Molecular peak in mass spectrum: m/e = 628

Intense peaks: 441, 351, 297, 145, 143.

Minor characteristic fragments: 585, 495, 323, 239.

f. In the same waydl-9α,15β-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid, preparedaccording to Preparations 6 and 8, was transformed into 9α,15β,18-trihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid and 9α,15β,19-trihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid.

The protected 18-hydroxy compound has:

C-value: 25.3

Molecular peak in mass spectrum: m/e = 628

Intense peaks: 441, 351, 297, 159, 143.

Minor characteristic fragments: 571, 481, 323, 239.

The protected 19-hydroxy product has:

C-value: 25.9

Molecular peak in mass spectrum: m/e = 628

Intense peaks: 441, 351, 297, 145, 143.

Minor characteristic fragments: 585, 495, 323, 239.

The fermentations with other Streptomyces species were all carried outaccording to the procedure described in Example III; the fermentationswith the other microorganisms were carried out according to theprocedure described in Example I.

EXAMPLE IV

a. Fermentation of dl-9β,15α-dihydroxy-prost-13(t)-enoic acid, preparedaccording to Preparations 4 and 8, with Ophiobolus graminis (ATCC 12761)yielded a small amount of 9β,15α,17 -trihydroxy-prost-13(t)-enoic acid.The main products of these fermentations were the corresponding 18- and19-hydroxy isomers, which were identical to the products of Example I g.

The silylated methyl ester of the 17-hydroxy compound has:

C-value: 23.7

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 223, 197, 145, 129.

Minor characteristic fragments: 543, 483, 453, 297, 259, 103.

b. In the same way dl-9-keto-15β-hydroxy-prost-13(t)-enoic acid,prepared according to the Preparations 3 and 8, when fermented withStreptomyces sp. (190.74) yielded a small amount of 9-keto-15β,17-dihydroxy-prost-13(t)-enoic acid, next to the 18- and 19-hydroxyisomers, which were identical to the products of Example I b.

The protected 17-hydroxy product (silyl ether, methyl ester, methoxime)has:

C-value: 25.3

Molecular peak in mass spectrum: m/e = 541

Intense peaks: 420, 382, 366, 250, 197, 145.

Minor characteristic fragments: 498, 438, 408, 259, 103.

c. In the same way dl-9α,15α-dihydroxy-prost-13(t)-enoic acid, preparedaccording to Preparations 4 and 8, when fermented with Streptomycesaureofaciens (ATCC 10762) yielded the 19-hydroxy derivative as the mainproduct and small amounts of the 18-hydroxy derivative and 9α,15α,17-trihydroxy-prost-13(t)-enoic acid as byproducts; the 18-hydroxy and19-hydroxy derivatives were identical to the products of Example I c.The silylated methyl ester of the 17-hydroxy compound has:

C-value: 23.7

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 297, 197, 145

Minor characteristic fragments: 543, 483, 453, 247, 103.

d. In the same way dl-9β,15β-dihydroxy-prost-13(t)-enoic acid, preparedaccording to Preparations 4 and 8, when fermented with Metarrhiziumbrunneum (CBS 316.51) yielded the 18- and 19-hydroxy derivatives as mainproducts (which were identical to the products of Example I d) and9β,15β,17 -trihydroxy-prost-13(t)-enoic acid as byproduct.

The silylated methyl ester of the 17-hydroxy compound has:

C-value: 23.7

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 223, 197, 145, 129.

Minor characteristic fragments: 543, 483, 453, 297, 259, 103.

e. In the same way dl-9α,15β-dihydroxy-prost-13(t)-enoic acid, preparedaccording to Preparations 4 and 8, when fermented with Streptomycesgriseus (CBS 479.48) yielded a small amount of 9α,15β,17-trihydroxy-prost-13(t)-enoic acid, next to the 18- and 19-hydroxyisomers, which were identical to the products of Example I h.

The silylated methyl ester of the 17-hydroxy compound has:

C-value: 23.7

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 297, 197, 145.

Minor characteristic fragments: 543, 483, 453, 247, 103.

EXAMPLE V

a. A culture of Stemphylium solani (NRRL 1805) was grown in a 10-10medium according to the procedure described in Example I a.

18 Hours after inoculation 20 mg ofdl-9α,15β-dihydroxy-prost-13(t)-enoic acid, prepared according toPreparations 4 and 8, dissolved in 2.5 ml of 50% aqueous ethanol wereadded and the incubation was continued for another 24 hours at 26° C.

According to TLC a new compound was formed which was more polar than thestarting material. The fermentation broth was filtered, the filtrateacidified to pH=3 with a 10% aqueous citric acid solution, and extractedthree times with 20 ml of ethyl acetate. The extract was evaporatedunder reduced pressure and the residue purified by column chromatography(SiO₂ pretreated with 1% acetic acid; eluted with ethyl acetate -heptane (8:3) containing 0.1% acetic acid). The matching fractions werecombined and evaporated under reduced pressure.

There was obtained 7 mg of 9α,15β,20-trihydroxy-prost-13(t)-enoic acid.The silylated methyl ester of this product has:

C-valve: 25.5

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 297, 129.

Minor characteristic fragments: 367, 197, 170, 142, 103.

b. In the same way dl-9β,15β-dihydroxy-prost-13(t)-enoic acid, preparedaccording to Preparations 4 and 8, was converted into9β,15β,20-trihydroxyprost-13(t)-enoic acid.

The silylated methyl ester of this product has:

C-valve: 25.5

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 129. Minor characteristic fragments: 367, 297,197, 170, 142, 103.

c. In the same way 9-keto-15α-hydroxy-prosta-5(c),8(12),13(t)-trienoicacid (PGB₂) was converted by Aspergillus niger (ATCC 9142) into9-keto-15α,20-dihydroxyprosta-5(c),8(12),13(t)-trienoic acid.

The protected product has:

C-valve: 28.5

Molecular peak in mass spectrum: m/e = 537

Intense peaks: 506, 416, 378, 162.

Minor characteristic fragments: 436, 246, 232, 184, 103.

d. In the same way 9-keto-15α-hydroxy-prosta-5(c),10,13(t)-trienoic acid(PGA₂) was transformed by Preussia fleischhakii (CBS 167.40) into9-keto-15α,20-dihydroxy-prosta-5(c),13(t)-dienoic acid.

The protected product has:

C-value: 27.1

Molecular peak in mass spectrum: m/e = 539

Intense peaks: 508, 129.

Minor characteristic fragments: 438, 380, 348, 226, 220, 198, 184, 142,103.

e. In the same waydl-9-keto-15α-hydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acid,prepared according to Preparations 7 and 8, was converted by Preussiafleischhakii (CBS 167.40) into 9-keto-15α,20 -dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acid.

The protected product has:

C-valve: 27.8

Molecular peak in mass spectrum: m/e = 583

Intense peaks: 396, 143, 131.

Minor characteristic fragments: 464, 462, 366, 364, 239, 144.

f. In the same way dl-9-keto-15β-hydroxy-15α-methyl-20-ethyl-prost-13(t)enoic acid, prepared according to Preparations 7 and 8, was transformedby Preussia fleischhakii (CBS 167.40) into 9-keto-15β,20-dihydroxy-15α-methyl-20 -ethyl-prost-13(t)-enoic acid.

The protected product has:

C-value: 27.8

Molecular peak in mass spectrum: m/e = 583

Intense peaks: 396, 142, 131.

Minor characteristic fragments: 464, 462, 366, 239.

g. In the same way dl-9α,15α-dihydroxy-20 ethyl-prost-13(t)-enoic acid,prepared according to Preparations 4 and 8, was converted into 9α,15α,20-trihydroxy-20 -ethyl-prost-13(t)-enoic acid.

The protected product has:

C-value: 26.3

Molecular peak in mass spectrum: m/e = 614

Intense peaks: 427, 337, 297, 246, 131, 129.

Minor characteristic fragments: 585, 495, 323, 310, 211.

h. In the same waydl-9α,15α-dihydroxy-15β-methyl-20-ethyl-prost-13(t)-enoic acid, preparedaccording to Preparations 6 and 8, was transformed into 9α,15α,20-trihydroxy-15β-methyl-20 -ethyl-prost-13(t)-enoic acid.

The protected product has:

C-value: 26.2

Molecular peak in mass spectrum: m/e = 628

Intense peaks: 441,351, 297, 143, 131.

Minor characteristic ffragments: 509, 419, 323, 239.

i. In the same waydl-9α,15β-dihydroxy-15α-methyl-20-ethyl-prost-13(t)-enoic acid, preparedaccording to Preparations 6 to 8, was converted into 9α,15β,20-trihydroxy-15α-methyl-20 -ethyl-prost-13 (t)-enoic acid.

The protected product has:

C-value: 26.3

Molecular peak in mass spectrum: m/e = 628

Intense peaks: 441, 351, 297, 143, 131.

Minor characteristic fragments: 509, 419, 323, 239.

j. In the same way dl-9-keto-15α-hydroxy-prost-13(t)-enoic acid,prepared according to Preparations 3 and 8, was converted by Pythiumultimum (CBS 296.37) into 9-keto-15α-20-dihydroxy-prost-13(t)-enoicacid.

The protected product has:

C-value: 27.2

Molecular peak in mass spectrum: m/e = 541

Intense peaks: 510, 382, 222, 129.

Minor characteristic fragments: 420, 368, 309, 197, 103.

k. In the same way dl-9-keto-15β-hydroxy-prost-13(t)-enoic acid,prepared according to Preparations 3 and 8, was converted by Curvulariatrifolli (CBS 210.59) into 9-keto-15β,20-dihydroxy-prost-13(t)-enoicacid.

The protected product has:

C-value: 27.4

Molecular peak in mass spectrum: m/e = 541

Intense peaks: 510, 382, 222, 129.

Minor characteristic fragments: 420, 368, 309, 197, 103.

l. In the same way dl-9β,15α-dihydroxy-prost-13(t)-enoic acid, preparedaccording to Preparations 4 and 8, was converted by Alternaria radicina(CBS 245.67) into 9β,15α,°-trihydroxy-prost-13(t)-enoic acid.

The protected product has:

C-value: 25.5

Molecular peak in mass spectrum: m/e = 586

Intense peaks: 427, 337, 129.

Minor characteriistic fragments: 313, 297, 197, 142, 103.

When the mold Delacroixia coronata (CBS 647.68) was fermented with thesubstrates mentioned in Examples V h and v i, the same 20-hydroxyderivatives were obtained, but as byproduct only. Main products withthis microorganism were then the 19-hydroxy derivatives of thesesubstrates.

EXAMPLE VI

a. 10 mg of 9-keto-11α,15α,18 -trihydroxy-prost-13(t)-enoic acid,prepared according to Example III a, were dissolved in 1 ml of methanol.To this solution 4 ml of an ethereal solution of diazomethane(containing 12 g of diazomethane per liter) were added. The reaction wasfollowed by thin layer chromatography (SiO₂, F₂₅₄ Merck; ethyl acetate /heptane / acetic acid / methanol / water = 40 / 20 / 4 / 6 / 3 ). After30 minutes the reaction was completed. The solvent was evaporated in astream of nitrogen and methyl 9-keto-11α,15α,18-trihydroxy-prost-13(t)-enoate was obtained as an oil.

b. 3.7 mg of 9-keto-11α,15α,19 -trihydroxy-prost-13(t)-enoic acid,prepared according to Example III a, were dissolved in 0.5 ml of ethylacetate. To the solution was added a solution of 1.5 mg oftriethanolamine in 0.5 ml of ethyl acetate. The resulting solution wasevaporated to dryness in a stream of nitrogen and then dried in vacuumto constant weight; 9-keto-11α,15α,19 -trihydroxy-prost-13(t)-enoic acidtriethanolamine salt obtained as an oil.

Other microorganisms capable of introducing an 18-, 19- or 20-hydroxygroup in the prostaglandin compounds of formula II are, for example:

Aspergillus amstelodami (CBS 521.65)

aspergillus chevalieri (CBS 414.67)

aspergillus flavus (CBS 178.74)

beauveria alba (CBS 348.55)

botryosphaeria rhodina (CBS 175.26)

botrytis cinerea (ATCC 12481)

coprinus bisporus (CBS 184.52)

corpinus congregatus (CBS 180.51)

cunninghamella blakesleeana (NRRL 1373)

cunninghamella echinulata (CBS 229.51)

curvularia ellisii (CBS 193.62)

diplodia alni (CBS 200.49)

drechslera buchloes (CBS 246.49)

Endothiella gyrosa Sacc. (CBS 253.54)

entomophtora virulenta (CBS 217.66)

fusarium semitectum (CBS 181.74)

fusarium ventricosum (CBS 205.31)

gliocladium viride Matr. (CBS 191.32)

gongronella butleri (CBS 259.52)

Hormodendrum chaquense (CBS 231.36)

hypomyces aurantius (CBS 207.29)

hypoxylon haematostroma (CBS 255.63)

hypoxylon jecorinum (CBS 258.63)

isoachlya turoloides (CBS 598.67)

lycoperdon gemmatum (CBS 182.74)

microascus cinereus (CBS 300.61)

microascus cirrosus (CBS 277.24)

microascus desmospours (CBS 424.62)

mycoacia stenodon (CBS 318.54)

nigrospora sacchari (CBS 290.62)

nodulisporium verrucosum (CBS 245.29)

paecilomyces cremeo-roseus (CBS 250.55)

paecilomyces farinosus (CBS 183.74)

pellicularia filamentosa (CBS 184.74)

pestalotia populi-nigrae (CBS 353.51)

petriella asymmetrica (CBS 297.58)

petriellidium boydii (CBS 593.73)

petriellidium ellipsoideum (CBS 418.73)

physalospora mutila (CBS 302.36)

physalospora rhodina (CBS 185.74)

pseudonectria pachysandricola (CBS 501.63)

rhizopus nigricans (ATCC 6227^(b))

Sepedonium chrysospermum (CBS 140.23)

septoria linicola (CBS 502.50)

sphaeropsis conspersa (CBS 209.25)

stemphylium consortiale (NRRL 2187)

Thielavia basicola (CBS 540.50)

Thielavia terricola (CBS 165.73)

verticillium lecanii (CBS 123.42)

Moreover, an 18- or 19-hydroxy group can also be introduced in theprostaglandin compounds of formula II by various species of the genusStreptomyces, for example the species:

Streptomyces chattanoogensis (ATCC 19673)

streptomyces chattanoogensis (ATCC 13358)

streptomyces natalensis (CBS 700.57)

and the species with the following CBS deposit numbers:

186.74, 187.74, 189.74, 190.74, 191.74, 192.74, 193.74, 193.74 and194.74.

We claim:
 1. The 18 - and 19 - hydroxy-prostaglandins, of the formula:##STR7## wherein the waved lines indicate that the substituents at therepresentative bonds are either in the α or β position; Z represents--CH₂ CH₂ -- or cis --CH=CH--; R₁ represents H, CH₃ -- or C₂ H₅ --; R₄is hydrogen or methyl and one of R' and R" is hydroxy and the other ishydrogen and pharmaceutically acceptable salts or the aliphatic estersthereof containing 1 to 5 carbon atoms.
 2. A compound according to claim1, which is 9-keto-11α,15α,18 -trihydroxy-prost-13(t)-enoic acid.
 3. Acompound according to claim 1, which is 9-keto-11α,15α,19-trihydroxy-prost-13(t)-enoic acid.
 4. A compound according to claim 1,which is 9-keto-11α,15α,18 -trihydroxy-prosta-5(c),13(t)-dienoic acid.5. A compound according to claim 1, which is 9-keto-11α,15α,19-trihydroxy-prosta-5(c),13(t)-dienoic acid.
 6. A compound according toclaim 1, which is methyl 9-keto-11α,15α,18-trihydroxy-prost-13(t)-enoate.
 7. A compound according to claim 1,which is 9-keto-11α,15α,19 -trihydroxy-prost-13(t)-enoic acidtriethanolamine salt.